Efficient synthesis of chelators for nuclear imaging and radiotherapy: compositions and applications

ABSTRACT

Novel methods of synthesis of chelator-targeting ligand conjugates, compositions comprising such conjugates, and therapeutic and diagnostic applications of such conjugates are disclosed. The compositions include chelator-targeting ligand conjugates optionally chelated to one or more metal ions. Methods of synthesizing these compositions in high purity are also presented. Also disclosed are methods of imaging, treating and diagnosing disease in a subject using these novel compositions, such as methods of imaging a tumor within a subject and methods of diagnosing myocardial ischemia.

This application claims the benefit of the filing date of U.S.Provisional Patent Application Ser. No. 60/828,347, filed Oct. 5, 2006,the entire contents of which are hereby incorporated by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the fields of chemicalsynthesis, imaging, radiotherapy, labeling, chemotherapy, medicaltherapy, treatment of cardiovascular disease and treatment of cancer.More particularly, the invention concerns novel methods of synthesizingchelator-targeting ligand conjugates. Organic methods of synthesis areset forth herein that yield chelator-targeting ligands of high purity incomparison to chelator-targeting conjugates prepared by aqueous methods.Methods of imaging a site using these conjugates, as well as kits forpreparing these conjugates, are also set forth herein. Methods ofdiagnosing and treating diseases (i.e., cancers, cardiovasculardiseases, infections and inflammation) in a subject using compositionsthat includes the aforementioned conjugates are also disclosed.

2. Description of Related Art

Biomedical imaging includes various modalities that are widely used byphysicians and researchers to assist with not only the diagnosis ofdisease in a subject, but also to gain a greater understanding of normalstructure and function of the body. Exemplary imaging modalities includePET, SPECT, gamma camera imaging, CT, MRI, ultrasound, dual imaging andoptical imaging.

In many instances, optimal imaging of a particular site within a subjectrequires the administration of a particular agent to the subject.Inorganic metals such as technetium (^(99m)Tc), iron, gadolinium,rhenium, manganese, cobalt, indium, platinum, copper, gallium, orrhodium have proved to be a valuable component of many imaging agents.

Labeling molecules with inorganic metals can be achieved by chelatingthe metal to combinations of oxygen, sulfur and nitrogen atoms, forexample, of particular compounds. Chelators such as sulfur colloid,diethylenetriaminepentaacetic acid (DTPA, O₄),ethylenediaminetetraacetic acid (EDTA, O₄) and1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid (DOTA, N₄)have been used for this purpose. However, inorganic metals that arechelated in this manner are of limited usefulness for imaging because oftheir fast clearance from the body.

The preferred radioactive label for imaging agents is technetium(^(99m)Tc) due to its favorable half life (6 hrs), ease of production,wide availability, low energy (140 keV) and low cost. The longerhalf-life of isotopes such as ^(99m)Tc facilitates shipping of theradiolabelled amino acids to hospitals without an on-site cyclotron ordedicated radiochemistry laboratory. However, attaching ^(99m)Tc todrugs for imaging purposes is often a challenge.

¹⁸⁸Re has good characteristics for imaging and for potential therapeuticuse because of its high β energy (2.1 MeV), short physical half-life(16.9 hr) and 155 keV gamma-ray emission for dosimetric and imagingpurposes. The short physical half-life of ¹⁸⁸Re allows for higher dosescompared with long-lived radionuclides. Furthermore, the short half-lifereduces the problems of radioactive waste handling and storage. Inparticular, ¹⁸⁸Re is available from an in-house generator system similarto a ^(99m)Tc generator. ¹⁸⁸Re can be obtained from a ¹⁸⁸W/¹⁸⁸Regenerator, which makes it very convenient for clinical use. Both^(99m)Tc and ¹⁸⁸Re emit gamma rays, so the dosimetry generated based on^(99m)Tc images is expected to be more accurate than that produced usingthe current standard radioisotope, Y-90.

Regarding imaging using positron emission tomography (PET), PETradiosynthesis must be rapid because the radioisotope will decay duringlengthy chemical synthesis and higher risk of radiation exposure mayoccur during radiosynthesis. Cyclotron-based tracers are constrained bythe availability of a local cyclotron and its high cost. The Food andDrug Administration (FDA) permits radiopharmaceutical production incentral commercial facilities under well-controlled conditions, anddistributes these to local clinics where they are administered.Similarly, radionuclide generator systems that can be produced in awell-controlled facility are embraced by current FDA procedures and havea long history of successful clinical application. A generator uses aparent-daughter nuclide pair wherein a relatively long-lived parentisotope decays to a short-lived daughter isotope that is used forimaging. The parent isotope, which is produced at a cyclotron facility,can be shipped to a clinical site and from which the daughter isotopemay be eluted on site for clinical use.

⁶⁸Ga has a high positron emitting quantity (89% of its total decay),therefore the main consideration with this radionuclide is its spatialresolution, which depends on the positron range (energy), thenon-colinearity of annihilating photons, intrinsic properties, size andgeometry of the detector and the selection of the reconstructionalgorithm. Aspects of the detector design, physical properties and theirinfluence on system spatial resolution have been extensively addressedby many authors, leading to a continuous optimization of hardware.Although the maximum positron energy of ⁶⁸Ga (max=1.90 MeV, mean=0.89MeV) is higher than that of ¹⁸F (max=0.63 MeV, mean=0.25 MeV), a studyusing Monte Carlo analysis on spatial resolution revealed that under theassumption of 3 mm spatial resolution of PET detectors, the conventionalfull width at half maximum (FWHM) of ¹⁸F and ⁶⁸Ga are indistinguishablein soft tissue (3.01 mm vs. 3.09 mm). It implies that with the spatialresolution at 5 to 7 mm of current clinical scanners, the imagingquality using ⁶⁸Ga-based tracers can be as good as that of ¹⁸F-basedagents and this has stimulated others to investigate potential⁶⁸Ga-based imaging agents. Further, ⁶⁸Ga-based PET agents possesssignificant commercial potential because the isotope can be producedfrom a ⁶⁸Ge generator (275-day half-life) on site and serve as aconvenient alternative to cyclotron-based PET isotopes, such as ¹⁸F or¹³N.

Regarding synthetic preparations of imaging agents, when such agents areprepared in aqueous (wet) conditions, purification of the agents cansometimes present a problem. Purification in aqueous conditions can beachieved using, for example, size exclusion chromatography, or dialysiswith membranes of particular molecular weight cut-offs; for example,dialysis is typically most effective when separating species ofmolecular weights of 1000 g/mol or higher. However, this method ofpurification often isolates not only the desired agent, but also anyother species that may pass through the membrane. Introduction ofimpurities into imaging agents may be problematic in future applicationsof the imaging agents, especially regarding imaging and/or therapeuticuses. For example, if an imaging agent incorporating a radionuclide (the“true” imaging agent) is thought to be pure but actually containsimpurities that also incorporate a radionuclide, the proper measurementor detection of the “true” imaging agent may be obscured or renderedfalse due to the presence of the impurities.

Methods of synthesizing organic compounds in organic media, which employorganic solvents and the use of protecting groups, typically offerimprovements in the purification of compounds over aqueouspurifications. The installation of protecting groups permits variousfunctional groups of intermediates during the synthesis to be protected,and facilitates the purification of those intermediates. Various meansof purification using organic solvents allow for separation andisolation of desired compounds, such as imaging agents, with very littleimpurities. Further, species of molecular weights under 1000 g/mol canoften easily be purified using organic chemistry purification methods.In view of the benefits offered by organic synthesis and purificationover aqueous purification, methods of organically synthesizing andpurifying imaging agents would likely yield agents of higher purity thanthose obtained via aqueous purification.

To date, certain imaging agents have been prepared only via aqueousmeans. The impurities present in these agents can detract from their useas imaging and/or therapeutic agents. Thus, a need exists for thepreparation of these and other agents using synthetic organic techniquesto allow for agents of higher purities to be obtained.

SUMMARY OF THE INVENTION

The present inventors have identified novel methods of synthesizingagents that are, in certain embodiments, conjugates of a chelator and atargeting ligand (also called a targeting moiety). Such agents may beused for imaging, diagnostic, and/or therapeutic purposes, for example.Both organic (solvent) and wet (aqueous) synthetic and purificationmethods are described, and it is shown that organic synthetic andpurification methods result in compounds of higher purity than thoseprepared/purified by wet chemistry. Compounds of high purity are bettercandidates for clinical application, for example. Furthermore, certaincompounds and methods of the present invention offer wide flexibilityand selectivity in terms of (1) available sites of conjugation of achelator to a targeting ligand and (2) atoms available for chelation toa metal ion.

Accordingly, one general aspect of the present invention contemplates amethod of synthesizing a chelator-targeting ligand conjugate comprising:

to at least one targeting ligand comprising at least one functionalgroup, wherein:

-   -   A, D, E and F are each independently H, lower alkyl, —COOH,        protected carboxylic acid, —NH₂, protected amine, thiol, or        protected thiol, wherein at least one position is —NH₂ or thiol;    -   B and C are each independently a secondary amine, a tertiary        amine, —S—, —S(O)—, or —S(O)₂—;    -   R₁, R₂, R₃ and R₄ are each independently H or lower alkyl;    -   X is selected from the group consisting of —CH₂—CH₂—,        —CH₂—CH₂—CH₂—, —CH₂—C(O)—, —C(O)—CH₂—, —C(O)—CH₂—CH₂— and        —CH₂—CH₂—C(O)—; and    -   the conjugation is between A, D, E or F of the chelator and at        least one unprotected functional group of each targeting ligand;

-   wherein at least one of A, D, E, F, or the targeting ligand    comprises a protected functional group, provided that at least one    functional group of the targeting ligand is unprotected, and    provided that when A and D are each —NH₂, neither B nor C is a    secondary or a tertiary amine. Conjugates of the present invention    may include one targeting ligand, or more than one targeting ligand.    In some embodiments, the conjugate includes two targeting ligands.    The targeting ligands may be identical, or may be of distinct types.    Types of targeting ligands are discussed in greater detail below.

Methods discussed herein are distinct from methods described incopending U.S. application Ser. No. 11/737,694, filed Apr. 19, 2007, andare distinct from methods described in copending InternationalApplication No. PCT/US2006/016784, filed May 4, 2006.

The chelator pictured above may also be visualized as the following:

In general, methods of the present invention take place in an organicmedium. As used herein, “organic medium” refers to solutions andpurification methods comprising one or more organic solvents. Solventchoices for the methods of the present invention will be known to one ofordinary skill in the art. Solvent choices may depend, for example, onwhich one(s) will facilitate the solubilizing of all the reagents, or,for example, which one(s) will best facilitate the desired reaction(particularly if the mechanism of the reaction is known). Solvents mayinclude, for example, polar solvents and/or non-polar solvents. Asolvent may be a polar aprotic solvent, such as dimethylsulfoxide.Solvents choices include, but are not limited to, dimethylformamide,dimethylsulfoxide, dioxane, methanol, ethanol, hexane, methylenechloride, tetrahydrofuran, and/or acetonitrile. In some embodiments,solvents include ethanol, dimethylformamide and/or dioxane. More thanone solvent may be chosen for any particular reaction or purificationprocedure. Water may also be admixed into any solvent choice; this canbe done to enhance the solubility of one or more reactants, for example.

In some embodiments, only the conjugation between a chelator and atargeting ligand takes place via organic synthesis (that is, in organicmedia). In some embodiments, only the synthesis of a chelator takesplace via organic synthesis. In some embodiments, only the chelation ofa valent metal ion takes place via organic synthesis. In certainembodiments, any one or more of these steps take place via organicsynthesis.

Any chelator (that is, a compound that is capable of chelating, orbinding, one or more metal ions) known to those of skill in the art maybe utilized using the methodology of the present invention, andexemplary chelators are described in further detail herein. Chelatorstypically bind to one or more metal ions via an ionic bond. In someembodiments, the chelator comprises DTPA (diethylenetriaminepentaaceticacid), one or more amino acids, or any combination of one or more ofthese groups. In certain embodiments, one or more amino acids areselected from the group consisting of glycine and cysteine. In someembodiments, the chelator is selected from the group consisting ofdicysteine, triglycine cysteine and tricysteine glycine. The number andchoices of amino acids may be limited by their solubility in organicmedia. In certain embodiments, the chelator is ethylenedicysteine (EC).

Targeting ligands are also described in further detail herein. While achelator may be conjugated (that is, chemically attached or bound) to atargeting ligand via any mode known to those of skill in the art (e.g.,a covalent bond, an ionic bond, a dative bond, an ion pair), typicallythe attachment comprises a covalent bond.

Methods of the present invention may further comprise at least onepurification step. Any compound of the present invention may be purifiedvia any method known to those of skill in the art. Persons of skill inthe art are familiar with such methods, and when those methods may beemployed. For example, in a multi-step synthesis that is aimed atarriving at a particular compound, a purification step may be performedafter every synthetic step, after every few steps, at various pointsduring the synthesis, and/or at the very end of the synthesis. In somemethods, one or more purification steps comprises technique selectedfrom the group consisting of silica gel column chromatography, HPLC(high-performance liquid chromatography) and LC (liquid chromatography).In certain embodiments, purification methods specifically exclude sizeexclusion chromatography and/or dialysis. Methods of purification aredescribed in more detail below.

In certain embodiments, unconjugated chelators and/or chelator-targetingligand conjugates are generated via synthetic organic methods in veryhigh purity relative to such compounds generated via aqueousmethodology. For example, in some embodiments of the present invention,an unconjugated chelator, an unprotected chelator, a protected chelator,a chelator-targeting ligand conjugate, or a metal ion-labeledchelator-targeting ligand conjugate generated via organic means (or anycompound comprising a combination of chelator, protecting group,targeting ligand and metal ion) is between about 90% and about 99.9%pure, compared to between about 50% and about 70% pure for the aqueousproduct. In certain embodiments, an unconjugated chelator, anunprotected chelator, a protected chelator, a chelator-targeting ligandconjugate, or a metal ion-labeled chelator-targeting ligand conjugategenerated via organic means (or any compound comprising a combination ofchelator, protecting group, targeting ligand and metal ion) is about orat least about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%,71%, 72%, 73%, 74%, 75%, 76, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.5%, 99.9% pure, or higher, or any range derivable therein. Incertain embodiments, the range is about 70% to about 99.9%. In certainembodiments, the range is about 75% to about 99.9%. In certainembodiments, the range is about 80% to about 99.9%. In certainembodiments, the range is about 85% to about 99.9%. In certainembodiments, the range is about 90% to about 99.9%. In certainembodiments, the range is about 95% to about 99.9%.

In certain embodiments of the present invention, at least one of A, D,E, or F is protected in at least one step using at least one protectingagent, and at least one functional group of the targeting ligand isprotected in at least one step using at least one protecting agent.Functional groups, as described herein, may be those of any type knownto one of skill in the art. The term “functional group” generally refersto how persons of skill in the art classify chemically reactive groups.Non-limiting examples include alkene, alkyne, aryl (e.g., phenyl,pyridinyl), alcohol, aldehyde, ketone, azide, halogen, ester, —COOH,—NH₂, thiol, a secondary amine, a tertiary amine, —S—, —S(O)— and—S(O)₂—. In some embodiments, at least one functional group comprises anatom selected from the group consisting of C, H, O, N, P and S.Positions A, B, C, D, E and/or F may comprise one or more functionalgroups (e.g., —COOH, —NH₂, thiol, a secondary amine, a tertiary amine,—S—, —S(O)—, or —S(O)₂—). In certain embodiments, at least onefunctional group of the targeting ligand comprises an atom selected fromthe group consisting of O, N, S and P. The functional group of thetargeting ligand may be, for example, selected from the group consistingof amino, amido, thiol, hydroxyl, ether, ester, carbonyl, carboxylicacid, sulfonamido, thioether, thioester and thiocarbonyl.

Both the targeting ligand and the chelator will typically have one ormore functional groups. Functional groups and protecting agents that maybe used to generate a protected functional group are described herein.Persons of skill in the art will understand that any functional groupmay be protected using a protecting agent as necessary, as describedherein. As such, a functional group may be protected (e.g., a protectedamine, such as —NH-Cbz) or unprotected—also called, “free” (such as—NH₂). As is known to those of skill in the art, protecting groups areutilized in organic syntheses and not aqueous syntheses.

Further, in certain embodiments, one or more protecting groups may beremoved. The removal of a protecting group can be done at any timeduring any method or synthesis described herein, but is typicallyperformed when the protecting group is no longer needed and thefunctional group that is being protected is desired to be “revealed.” Inany method described herein, any compound comprising a chelatordescribed herein (e.g., a chelator-targeting ligand conjugate, a metalion labeled-chelator-targeting ligand conjugate) may not contain anyprotecting groups, or may comprise one or more protecting groups. Forexample, a site may be imaged using a metal ionlabeled-chelator-targeting ligand conjugate that contains no protectinggroups, or contains one or more protecting groups.

In certain embodiments, the targeting ligand comprises a leaving group.The term “leaving group” generally refers to groups readily displaceableby a nucleophile, such as an amine, an alcohol or a thiol nucleophile.Such leaving groups are well known and include, for example,carboxylates, N-hydroxysuccinimide, N-hydroxybenzotriazole, halides,triflates, tosylates, mesylates, alkoxys, thioalkoxys, sulfonyls and thelike.

In further embodiments, the three or more functional groups of thechelator together form a chelate. Typically, three or four atomstogether form a chelate. In certain embodiments, the chelate is selectedfrom the group consisting of NS₂, N₂S, S₄, N₂S₂, N₃S and NS₃. Forexample, three thioethers and one secondary amine may form an NS₃chelate. In some embodiments, such as with ethylenedicysteine, thechelate is an N₂S₂ chelate. Chelates may be that of any type known tothose of skill in the art, and are further described herein. Other atomsbesides N and S may comprise a chelate, such as oxygen.

As used herein, “chelate” may be used as a noun or a verb. As a noun,“chelate” refers to one or more atoms that are either capable ofchelating one or more metal ions, or are chelating to one or more metalions. Metal ions are described in more detail herein. In someembodiments, only one metal ion coordinates to a chelate. A non-limitingexample of “chelate” includes “an N₂S₂” chelate: this means that twonitrogen atoms and two sulfur atoms of a chelator are either a) capableof chelating to one or more metal ions or b) are coordinated to (orchelated to) to one or more metal ions. Accordingly, in someembodiments, the chelate is N₂S₂. A compound comprising a chelate is achelator. Typically, just one metal ion is chelated to a chelator.

In certain embodiments, at least one of A, D, E and F is a thiol. Thethiol may be protected in at least one step using at least one thiolprotecting agent. The thiol protecting agent may be any of those knownto those of skill in the art. For example, the thiol protecting agentmay be selected from a group consisting of an alkyl halide, a benzylhalide, a benzoyl halide, a sulfonyl halide, a triphenylmethyl halide, amethoxytriphenylmethyl halide and cysteine.

In certain embodiments, at least one of A, D, E and F comprises aprimary amine or at least one of B and C comprises a secondary amine. Incertain embodiments, at least one amine may be protected in one or moresteps using at least one amine protecting agent. Amine protecting agentsmay be any of those known to those of skill in the art. For example, theamine protecting group may be selected from the group consisting ofbenzylchloroformate, p-nitro-chlorobenzylformate, ethylchloroformate,di-tert-butyl-dicarbonate, triphenylmethyl chloride andmethoxytriphenylmethyl chloride.

In certain embodiments, the chelator is ethylenedicysteine. Whenemploying ethylenedicysteine as a chelator in the synthesis of anethylenedicysteine-targeting ligand conjugate, the two thiol groups ofethylenedicysteine are protected using at least one thiol protectingagent (e.g., using two or more equivalents of a thiol protecting agent)and in another step the two amine groups of ethylenedicysteine areprotected using at least one amine protecting agent (e.g., using two ormore equivalents of an amine protecting agent). Since thiol groups aremore reactive than amine groups, thiol groups will typically beprotected before amine groups are protected when both are initiallyunprotected (“free”).

As mentioned, conjugation between the chelator and a targeting ligandmay take place via any method and chemical linkage known to those ofskill in the art. That is, the targeting ligand may be conjugated orbound to one or more chelators in any manner known to those of ordinaryskill in the art. In certain embodiments, conjugation between thechelator and the targeting ligand takes place in a single step (i.e., a“one-pot” reaction). As is known by those of skill in the art, suchone-step reactions are desirable as they save time, help minimize wastereagents and minimize loss of product. Any of A, B, C, D, E, and/or Fmay participate in conjugation to a targeting ligand. In addition, anyof A, B, C, D, E, and/or F may participate in chelation. Further, any ofA, B, C, D, E, and/or F may participate in both chelation andconjugation. Such flexibility allows chelators of the present inventionto be manipulated in a variety of ways, depending on, for example, thereactivity of a chosen targeting ligand, the selectivity of conjugationdesired, the solubility of the reagents, the metal ion desired forchelation, etc. Typically, but not always, conjugation occurs prior tochelation.

Typically, one type of targeting ligand is conjugated to one chelator,but multiple targeting ligands may be conjugated to a single chelator.Commonly, during the organic synthesis of chelator-targeting ligandconjugates, as between the chelator and the targeting ligand, one actsas the nucleophile and one acts as the electrophile such thatconjugation takes place via a covalent bond. The covalent bond may be ofany type known to those of skill in the art. In some embodiments, thecovalent bond is selected from the group consisting of an amide bond, anether bond, an ester bond, a thioether bond, a thioester bond, asulfonamido bond and a carbon-carbon bond. The carbon-carbon bond istypically a single bond, but can also be a double or a triple bond. Whenacting as electrophiles, chelators and targeting ligands may comprisefunctional groups such as halogens and sulfonyls, which act as leavinggroups during conjugation. In some embodiments, the conjugation takesplace at one or more functional groups of the chelator selected from thegroup consisting of carboxylic acid, amine and thiol. Targeting ligandsmay also comprise nucleophilic groups, such as —NH₂, which mayparticipate in conjugation with an electrophilic chelator. Modes ofconjugation are discussed in greater detail below.

In certain embodiments, the chelator-targeting ligand conjugate furthercomprises a linker between the chelator and the targeting ligand. Such alinker may, for example, provide for easier conjugation between thechelator and the targeting ligand by providing a reactive group thatfacilitates the conjugation reaction. The linker may be of any typeknown to those of skill in the art. The linker may be initially attachedto the chelator or to the targeting ligand. A linker may be attached tothe chelator, while another linker is attached to the targeting ligand,such that the two linkers may then be joined. Persons of skill in theart will be familiar with the types of linkers available for methods ofthe present invention. In some embodiments, the linker is selected fromthe group consisting of a peptide, glutamic acid, aspartic acid, bromoethylacetate, ethylene diamine, lysine and any combination of one ormore of these groups.

In certain embodiments, E and F are each independently selected from thegroup consisting of —COOH, —NH₂, or thiol. In some embodiments, E and Fare each —COOH. In certain embodiments, the conjugation of at least onetargeting ligand takes place at E and/or F. In certain embodiments, eachof A and D are each protected by at least one protecting group prior toconjugation.

As one of skill in the art would appreciate, in order to conjugate achelator to a targeting ligand, at least one functional group of thechelator and at least one functional group of the targeting ligand mustbe “free” (that is, unprotected by a protecting group) such that the twocompounds may be joined together.

The chelator may further comprise a spacer, X. In certain aspects, useof a spacer allows for the proper number and orientation of chelatingatoms to chelate a metal ion. Persons of skill in the art will befamiliar with the types of spacers that may be used for methods of thepresent invention, and examples of spacers are disclosed below. Forexample, an alkyl spacer, such as (—CH₂—)_(n), wherein n is 1-100, maybe employed. One type of chelator employable in methods of the presentinvention that comprises an ethylene spacer is ethylenedicysteine (EC).In certain embodiments, X is —CH₂—C(O)—, —C(O)—CH₂—, —CH₂—CH₂—C(O)—, or—C(O)—CH₂—CH₂— and B and/or C is a secondary amine. This embodimenttypically results in either B or C being less nucleophilic than theother. For example, if together B, C and L are depicted as—NH—C(O)—CH₂—CH₂—NH—, the secondary amine of position C will be morenucleophilic than the secondary amine of B. Thus, C will be morereactive, resulting in selective conjugation of a targeting ligand atposition C. In certain embodiments, both positions A and D or E and Fare each protected by at least one protecting group prior to conjugationat C.

One feature of using amide bonds, such as when B, C, and L together form—NH—C(O)—CH₂—CH₂—NH—, lies in the fact that reactions wherein a metalion is chelated to a chelator often take place in acidic media. Amidebonds are relatively resistant to degradation in acidic media, andtherefore provide structural stability in the chelator during suchchelation reactions. Thus, X together with B and/or C may comprise anamide bond.

Chelator-targeting ligand conjugates chelated to a metal ion mayfunction as, for example, imaging and/or diagnostic agents, as describedherein. They can also function as therapeutic agents, or agents for dualdiagnosis and therapy, or dual imaging and therapy. Accordingly, incertain embodiments, methods of the present invention further comprisechelation of a metal ion to a chelator to generate a metal ionlabeled-chelator-targeting ligand conjugate. The metal ion may be any ofthose known to one of ordinary skill in the art. The metal ion may be a“cold” (non-radioactive) metal ion, or a radionuclide. In non-limitingexamples, the metal ion may be selected from the group consisting of atechnetium ion, a copper ion, an indium ion, a thallium ion, a galliumion, an arsenic ion, a rhenium ion, a holmium ion, a yttrium ion, asamarium ion, a selenium ion, a strontium ion, a gadolinium ion, abismuth ion, an iron ion, a manganese ion, a lutetium ion, a cobalt ion,a platinum ion, a calcium ion and a rhodium ion. The cold metal ion maybe, for example, selected from the group consisting of Cu-62, As-72,Re-187, Gd-157, Bi-213, Fe-56, Mn-55, an iron ion, a manganese ion, acobalt ion, a platinum ion and a rhodium ion.

The metal ion may be a radionuclide, and may be any radionuclide knownto those of skill in the art. The radionuclide, in some embodiments, maybe selected from the group consisting of ^(99m)Tc, ¹⁸⁸Re, ¹⁸⁶Re, ¹⁵³Sm,¹⁶⁶Ho, ⁹⁰Y, ⁸⁹Sr, ⁶⁷Ga, ⁶⁸Ga, ¹¹¹In, ¹⁴⁸Gd, ⁵⁵Fe, ²²⁵Ac, ²¹²Bi, ²¹¹At,⁴⁵Ti, ⁶⁰Cu, ⁶¹Cu, ⁶⁷Cu, and ⁶⁴Cu. In some embodiments, the metal ion is^(99m)Tc.

If the metal ion is chosen to be ^(99m)Tc, for example, the method mayfurther comprise the addition of a reducing agent. The reducing agentmay be that of any known to those of skill in the art. In someembodiments, the reducing agent comprises an ion selected from the groupconsisting of a dithionite ion, a stannous ion and a ferrous ion. Insome embodiments, the metal ion is ¹⁸⁸Re. In other embodiments, themetal ion is ⁶⁸Ga.

When a metal ion is employed in the method of the present invention, themetal ion may be chelated to any chelate known to those of skill in theart, as described herein. Persons of skill in the art recognize thatmetal ions chelate to varying numbers of atoms depending on, forexample, the type of metal, its valency and the atoms available forchelation. For example, three or four atoms of the chelator may chelateto one metal ion. In certain embodiments, a chelated metal ion may be^(99m)Tc. In certain embodiments, a chelated metal ion may be ¹⁸⁶Re. Incertain embodiments, a chelated metal ion may be ¹⁸⁷Re.

In some embodiments, the chelate may be selected from the groupconsisting of NS₂, N₂S, S₄, N₂S₂, N₃S and NS₃. In certain embodiments,any one or more of these chelates may not be a chelate of the presentinvention. In some embodiments, N₃S is not a chelate. In certainembodiments, the chelate is N₂S₂, for example, ethylenedicysteine.Methods of the present invention may further comprise the synthesis of ametal ion labeled-chelator-targeting ligand conjugate wherein thetargeting ligand participates with A, B, C, D, E, and/or F in chelationto a metal ion. Metal ions, chelation and targeting ligands arediscussed in more detail below. In some embodiments, the metal ion canbe imaged. The imaging can be by any method known to those of ordinaryskill in the art. Exemplary methods of imaging are discussed at lengthin the specification below, and include PET and SPECT.

As discussed above, metal ion labeled-chelator-targeting ligandconjugates prepared via organic synthesis typically enjoy puritieshigher than those achieved via aqueous preparations. For example, insome embodiments of the present invention, the metal ionlabeled-chelator-targeting ligand conjugate generated via organic meansis between about 90% and about 99.9% pure, compared to between about 50%and about 70% pure for the aqueous product. In certain embodiments, themetal ion labeled-chelator-targeting ligand conjugate synthesized viaorganic means is about or at least about 60%, 61%, 62%, 63%, 64%, 65%,66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% pure, or higher, or any rangederivable therein.

Any chelator described herein may be chelated to a metal ion. Aprotected chelator may be used, or an unprotected chelator. The chelatormay be chelated before or after the chelator is purified.

In certain embodiments, generation of a metal ionlabeled-chelator-targeting ligand conjugate comprises:

-   -   (a) removing at least one protecting group from a        chelator-targeting ligand conjugate as described herein; and    -   (b) chelating a metal ion to the chelator of the        chelator-targeting ligand conjugate.

In certain embodiments, generation of a metal ionlabeled-chelator-targeting ligand conjugate comprises:

-   -   (a) obtaining a chelator of the following formula:

-   -   wherein A, B, C, D, E, F, X, R₁, R₂, R₃ and R₄ are as defined        above and at least one of A, B, C, D, E, F, X, R₁, R₂, R₃ and R₄        is protected by at least one protecting agent;    -   (b) conjugating the chelator to a targeting ligand to generate a        chelator-targeting ligand conjugate;    -   (c) removing at least one protecting group from the        chelator-targeting ligand conjugate; and    -   (d) chelating a metal ion as described herein to the chelator of        the chelator-targeting ligand conjugate.

Indeed, it is contemplated that any compound described herein comprisingone or more protecting groups may, in any particular method, undergoremoval of one or more protecting groups. A protecting group may beremoved, for example, from the chelator moiety, the targeting ligandmoiety, or both moieties in one or more steps before or after achelator-targeting ligand conjugate is chelated to a metal ion, asdescribed herein. Protecting groups are described in more detail herein,including their installation and removal.

In other embodiments, generation of a metal ionlabeled-chelator-targeting ligand conjugate comprises:

-   -   (a) chelating a metal ion to a chelator as described herein to        generate a metal ion labeled-chelator;    -   (b) conjugating the metal ion labeled-chelator to a targeting        ligand; and    -   (c) removing one or more protecting groups from the metal ion        labeled-chelator-targeting ligand conjugate.

Certain embodiments of the present invention contemplate a method ofsynthesizing a protected chelator comprising:

(a) obtaining a chelator of the following formula:

wherein:

-   -   A, D, E and F are each independently H, lower alkyl, —COOH,        —NH₂, or thiol, wherein at least one position is —COOH, —NH₂, or        thiol;    -   B and C are each independently a secondary amine, a tertiary        amine, —S—, —S(O)—, or —S(O)₂—;    -   R₁, R₂, R₃ and R₄ are each independently H or lower alkyl; and    -   X is selected from the group consisting of —CH₂—CH₂—,        —CH₂—CH₂—CH₂—, —CH₂—C(O)—, —C(O)—CH₂—, —C(O)—CH₂—CH₂— and        —CH₂—CH₂—C(O)—; and

(b) protecting the —COOH, —NH₂, or thiol using a carboxylic acidprotecting agent, an amine protecting agent, or a thiol protectingagent, respectively.

As for any synthetic method of the present invention, the method may becarried out in an organic medium. The protected chelator may beprotected ethylenedicysteine. The method may further comprise apurification step, a chelation step comprising chelation of a metal ion,the removal of at least one protecting group, or any combination ofthese steps. (Indeed, any method described herein may comprise apurification step, a chelation step comprising chelation of a metal ion,the removal of at least one protecting group, or any combination ofthese steps.) In this or any method described herein, the protectedchelator may be about 80% to about 99.9% pure. For example, theprotected chelator may be about 80% to about 90% pure. In this or anymethod described herein comprising a chelator with the core structureshown above, when A and D are each —NH₂, neither B nor C may be asecondary or a tertiary amine.

Certain embodiments of the present invention also contemplate achelator-targeting ligand conjugate of the following formula:

wherein:

-   -   A, D, E and F each independently comprise H, lower alkyl, —COOH,        a protected carboxylic acid, —NH₂, a protected amine, thiol, a        protected thiol, an unprotected targeting ligand, or a protected        targeting ligand,        -   wherein at least one of A, D, E and F comprises a protected            carboxylic acid, a protected amine, or a protected thiol and            at least one of A, D, E and F comprises a protected            targeting ligand or an unprotected targeting ligand;    -   B and C are each independently a secondary amine, a tertiary        amine, —S—, —S(O)—, or —S(O)₂—;    -   R₁, R₂, R₃ and R₄ are each independently H or lower alkyl;    -   X is selected from the group consisting of —CH₂—CH₂—,        —CH₂—CH₂—CH₂—, —CH₂—C(O)—, —C(O)—CH₂—, —C(O)—CH₂—CH₂— and        —CH₂—CH₂—C(O)—; and    -   wherein the chelator-targeting ligand conjugate is between about        70% and about 99.9% pure.        The conjugate may be between about 80% and about 99.9% pure. The        conjugate may be between about 90% and about 99.9% pure. The        conjugate may be further defined as a metal ion        labeled-chelator-targeting ligand conjugate. The conjugate may        be further defined as ^(99m)Tc-EC-glucosamine,        ¹⁸⁶Re-EC-glucosamine, or ¹⁸⁷Re-EC-glucosamine.

As mentioned, the targeting ligand may be of any type known to those ofskill in the art, and such ligands are discussed in more detail herein.A “targeting ligand” is defined herein to be a molecule or part of amolecule that binds with specificity to another molecule. One ofordinary skill in the art would be familiar with the numerous agentsthat can be employed as targeting ligands in the context of the presentinvention. The targeting ligand can be any such molecule known to thoseof ordinary skill in the art. Non-limiting examples of targeting ligandsinclude a tissue-specific ligand, an antimicrobial, an antifungal, or animaging agent.

In some embodiments, the targeting ligand is a “tissue-specific ligand.”A “tissue-specific ligand” is defined herein to refer to a molecule or apart of a molecule that can bind or attach to one or more tissues. Thebinding may be by any mechanism of binding known to those of ordinaryskill in the art.

Non-limiting examples of tissue-specific ligands include a drug, a DNAtopoisomerase inhibitor, a DNA intercalator, an antimetabolite, adisease cell cycle targeting compound, a gene expression marker, anangiogenesis targeting ligand, a tumor marker, a folate receptortargeting ligand, an apoptotic cell targeting ligand, a hypoxiatargeting ligand, a disease receptor targeting ligand, a receptormarker, a peptide, a nucleotide, an antibody, an antisense molecule, asiRNA, glutamate pentepeptide, an agent that mimics glucose, amifostine,angiostatin, monoclonal antibody C225, monoclonal antibody CD31,monoclonal antibody CD40, capecitabine, deoxycytidine, fullerene,herceptin, human serum albumin, lactose, quinazoline, thalidomide,transferrin, and trimethyl lysine.

In some embodiments, the tissue-specific ligand may be a drug, such asan anticancer agent. Non-limiting examples of anti-cancer agents includetamoxifen, topotecan, LHRH, podophyllotoxin, colchicine, endostatin,tomudex, thiotepa, cyclosphosphamide, busulfan, improsulfan, piposulfan,benzodepa, carboquone, meturedepa, uredepa, altretamine,triethylenemelamine, trietylenephosphoramide,triethiylenethiophosphoramide, trimethylolomelamine, bullatacin,bullatacinone, bryostatin, callystatin, CC-1065, adozelesin, carzelesin,bizelesin, cryptophycin 1, cryptophycin 8, dolastatin, duocarmycin,KW-2189, CB1-TM1, eleutherobin, pancratistatin, a sarcodictyin,spongistatin, chlorambucil, chlomaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard, carmustine, chlorozotocin, fotemustine,lomustine, nimustine, and ranimustine, calicheamicin, dynemicin,clodronate, an esperamicin, neocarzinostatin chromophore, anaclacinomysin, actinomycin, authramycin, azaserine, a bleomycin,cactinomycin, carabicin, caminomycin, carzinophilin, a chromomycin,dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine,epirubicin, esorubicin, idarubicin, marcellomycin, mycophenolic acid,nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin,quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,ubenimex, zinostatin, zorubicin, 5-fluorouracil (5-FU), denopterin,methotrexate, pteropterin, trimetrexate, 6-mercaptopurine, thiamiprine,thioguanine, ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine,calusterone, dromostanolone propionate, epitiostanol, mepitiostane,testolactone, aminoglutethimide, mitotane, trilostane, folinic acid,aceglatone, aldophosphamide glycoside, aminolevulinic acid, eniluracil,amsacrine, bestrabucil, bisantrene, edatraxate, defofamine, demecolcine,diaziquone, elformithine, elliptinium acetate, an epothilone, etoglucid,gallium nitrate, hydroxyurea, lentinan, lonidainine, a maytansinoid,mitoguazone, mopidanmol, nitraerine, pentostatin; phenamet; pirarubicin;losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine, PSKpolysaccharide complex, razoxane, rhizoxin, sizofuran, spirogermanium,tenuazonic acid, triaziquone, 2,2′,2″-trichlorotriethylamine, atrichothecene, urethan, vindesine, dacarbazine, mannomustine,mitobronitol, mitolactol, pipobroman, gacytosine, arabinoside (“Ara-C”),cyclophosphamide, thiotepa, doxetaxel, chlorambucil, 6-thioguanine,mercaptopurine, methotrexate, cisplatin, oxaliplatin, carboplatin,vinblastine, platinum, ifosfamide, mitoxantrone, vincristine,vinorelbine, novantrone, teniposide, edatrexate, daunomycin,aminopterin, xeloda, ibandronate, irinotecan, RFS 2000,difluoromethylornithine (DMFO), retinoic acid, and capecitabine.

Other examples of drugs include cardiovascular drugs. Non-limitingexamples of such drugs include an antihyperlipoproteinemic agent, anantiarteriosclerotic agent, an antithrombotic agent, a fibrinolyticagent, an antiplatelet agent, a blood coagulant, a thrombolytic agent,an antiarrythmic agent, an antihypertensive agent, a vasopressor, ananti-angiotension II agent, an afterload-preload reduction agent, adiuretic, and an inotropic agent. Examples of cardiovascular drugsinclude mexiletine, tocamide, moricizine, procainamide, diisopyramide,quinidine, popafenone, flecaimide, encamide, bepridil, verapamil,diltiazem, bretylium, sotalol, amiodarone, ibutilide, propranolol,atropine, adenosine and digoxin. More examples are set forth below.

In some embodiments, the targeting ligand is a DNA topoisomeraseinhibitor. Non-limiting examples include a fluoroquinolone antibiotic,irinotecan, topotecan, etoposide, teniposide, lurtotecan, exatecan andrubitecan. Non-limiting examples of DNA intercalators include7-aminoactinomycin, ethidium, proflavin, daunomycin, doxorubicin, andthalidomide.

In some embodiments, the targeting ligand is an antimetabolite.Non-limiting examples include azathioprine, a mercaptopurine, apyrimidine, a sulfanilamide drug, methotrexate, tetrahydrofolate, folicacid, pemetrexed, raltitrexed, thioguanine, fludarabine, pentostatin,cladribine, fluorouracil, floxuridine, and gemcitabine.

The targeting ligand may be a disease cell cycle targeting ligand.Non-limiting examples include adenosine, FIAU, FIRU, IVFRU, GCV, PCV,FGCV, FPCV, FHPG, FHBG and guanine.

In some embodiments, the targeting ligand is a gene expression marker.For example, the gene expression marker may be an epidermal growthfactor receptor ligand. In further embodiments, the targeting ligand isan angiogenesis targeting ligand. Non-limiting examples include a COX-2inhibitor, anti-EGF receptor, herceptin, angiostatin, or thalidomide.Examples of COX-2 inhibitors include celecoxib, rofecoxib, andetoricoxib.

Other examples of targeting ligands include tumor markers. Non-limitingexamples of tumor markers include PSA, ER, PR, CA-125, CA-199, CEA, AFP,an interferon, BRCA1, HER-2/neu, cytoxan, p53 and endostatin. Thetargeting ligand may also be a folate receptor targeting ligand.Examples include folate, methotrexate and tomudex.

The targeting ligand may also be an apoptotic cell targeting ligand. Forexample, the apoptotic cell targeting ligand may further be defined as atumor apoptotic cell targeting ligand. Non-limiting examples include aTRAIL monoclonal antibody, a substrate of caspase-3 and a Bcl familymember. Examples of a substrate of caspase-3 include a peptide orpolypeptide comprising the amino acid sequence aspartic acid-glutamicacid-valine-aspartic acid. Examples of Bcl family members include Bax,Bcl-xL, Bid, Bad, Bak and Bcl-2

In some embodiments, the targeting ligand is a hypoxia targeting ligand.For example, the hypoxia targeting ligand may be a tumor hypoxiatargeting ligand, a cardiac ischemia marker, a cardiac viability tissuemarker, a congestive heart failure marker, or a rest/stress cardiactissue marker. Non-limiting examples of tumor hypoxia targeting ligandsinclude annexin V, colchicine, a nitroimidazole, mitomycin,metronidazole, 99 mTc-HL91, and Cu-ATSM. Non-limiting examples ofcardiac ischemia markers include interleukin-6, tumor necrosis factoralpha, matrix metalloproteinase 9, myeloperoxidase, intercellular andvascular adhesion molecules, soluble CD40 ligand, placenta growthfactor, high sensitivity C-reactive protein (hs-CRP), ischemia modifiedalbumin (IMA), free fatty acids, and choline. Non-limiting examples ofcardiac viability tissue markers include phospholipase C, myosinlight-chain phosphatase, nitric oxide, prostacyclin, endothelin,thromboxane, L-arginine and L-citrulline. Non-limiting examples ofcongestive heart failure markers include interleukin-1, cardiotrophin-1,insulin-like growth factor, epidermal growth factor, tyrosine kinasereceptor, angiotensin II, and metronidazole. Non-limiting examples ofrest/stress cardiac tissue markers include a mitogen-activated proteinkinase, cyclic adenosine monophosphate, phospholipase C,phosphatidylinositol bisphosphate, isositol trisphosphate,diacylglycerol, a tyrosine kinase, and metronidazole.

Non-limiting examples of peptides contemplated as targeting ligandsinclude neuropeptide Y, calcitonin gene-related peptide, substance P,and vasoactive intestinal peptide. Non-limiting examples of nucleotidescontemplated as targeting ligands include adenine, thymine, guanine,cytosine, and uracil. Non-limiting examples of antibodies contemplatedas targeting ligands include an antibody that binds to a troponin,tropomyosin, a sarcolemmal, a collagen, a matrix metalloproteinase, or atissue inhibitor of a matrix metalloproteinase.

In some embodiments, the targeting ligand is an antisense molecule or ansiRNA. The targeting ligand may also be glutamate pentapeptide.

In particular embodiments, the targeting ligand is an agent that mimicsglucose. Non-limiting examples of agents that mimic glucose includedeoxyglucose, glucosamine, tetraacetylated glucosamine, neomycin,kanamycin, gentamycin, paromycin, amikacin, tobramycin, netilmicin,ribostamycin, sisomicin, micromicin, lividomycin, dibekacin, isepamicin,astromicin and aminoglycoside. In particular embodiments, the agent thatmimics glucose is glucosamine.

In further embodiments, the targeting ligand is a disease receptortargeting ligand. Non-limiting examples of disease receptor targetingligands include an estrogen, an androgen, luteinizing hormone,luteinizing hormone releasing hormone (LHRH), transferrin, a progestin,tetraacetate mannose, α-β-tyrosine, tyrosine, a tyrosine derivative,estrone, tamoxifen, and α-methyltyrosine.

Other general aspects of the present invention contemplate a compositioncomprising a metal ion labeled-chelator-targeting ligand conjugatesynthesized by any of the methods described herein. In particularembodiments, the metal ion labeled-chelator-targeting ligand conjugatecomprises ethylenedicysteine chelated to a metal ion selected from thegroup consisting of ^(99m)Tc, ⁶⁸Ga ¹⁸⁸Re, ¹⁸⁷Re and ¹⁸⁶Re; the targetingligand comprises a ligand selected from the group consisting ofglucosamine, deoxyglucose, metronidazole, annexin V, guanine and LHRH;and the conjugation between the chelator and the targeting ligand takesplace via an amide bond or an ester bond.

Exemplary anti-cancer compositions include a chelator capable ofchelating to a therapeutic radiometallic substance, such as Re-188,Re-187, Re-186, Ho-166, Y-90, Sr-89, or Sm-153, arsenic, cobalt, copper,calcium, selenium, thallium or platinum. Other exemplary anti-cancerligands include, for example, epipodophyllotoxin, vincristine,docetaxel, paclitaxel, daunomycin, doxorubicin, mitoxantrone, topotecan,bleomycin, gemcitabine, fludarabine and 5-FUDR. In certain particularembodiments, the anti-cancer ligand is methotrexate.

Other aspects of the present invention contemplate a compositioncomprising a chelator-targeting ligand conjugate synthesized by any ofthe methods described herein. In certain embodiments, the inventioncontemplates a composition comprising a metal ionlabeled-chelator-targeting ligand conjugate synthesized by any of themethods described herein. In any given composition embodiment, thechelator-targeting ligand conjugate composition may comprise one or moreprotecting groups at any position of either/both the chelator and/or thetargeting ligand, or no protecting groups at all. Furthermore, thechelator or chelator-targeting ligand conjugate may or may not comprisea metal ion.

Embodiments of the present invention also pertain to a compositioncomprising a metal ion-labeled chelator-targeting ligand conjugatesynthesized by any of the methods set forth herein. The composition mayinclude a pharmaceutically acceptable carriers such as glutamic acid andothers mild acids and cold metals. In some embodiments, the compositioncomprises (a) the metal ion labeled-chelator-targeting ligand conjugatecomprises ethylenedicysteine chelated to a metal ion selected from thegroup consisting of ^(99m)Tc, ⁶⁸Ga, ¹⁸⁸Re, and ¹⁸⁷Re; (b) the targetingligand comprises a ligand selected from the group consisting ofglucosamine, deoxyglucose, metronidazole, annexin V, guanine and LHRH;and (c) the conjugation between the chelator and the targeting ligandtakes place via an amide bond or an ester bond.

Further embodiments of the present invention include a reagent forpreparing an imaging agent, a therapeutic agent or a radio/therapeuticagent, comprising a metal ion labeled-chelator conjugate prepared by anyof the methods set forth herein. In specific embodiments, the reagent isa reagent for preparing a chemotherapeutic agent or aradio/chemotherapeutic agent. In some embodiments, the metal ionlabeled-chelator-targeting ligand conjugate is between about 90% andabout 99.9% pure. In certain embodiments, the metal ionlabeled-chelator-targeting ligand conjugate comprisesethylenedicysteine.

The present invention also pertains to kits for preparing an imagingagent, a therapeutic agent, or a radio/therapeutic agent, comprising oneor more sealed containers and a predetermined quantity of a compositioncomprising a chelator-targeting ligand conjugate prepared by any methoddescribed herein in one or more sealed containers. In some embodiments,the kit includes a chelator-targeting ligand conjugate that is betweenabout 90% and about 99.9% pure. In some embodiments, the kit includes achelator-targeting ligand conjugate that is between about 80% and about99.9% pure. In some embodiments, the kit includes a chelator-targetingligand conjugate that is between about 70% and about 99.9% pure. Inparticular embodiments, the kit includes an ethylenedicysteine-targetingligand conjugate. In some embodiments, the kit further includes a metalion. The metal ion may or may not be a radionuclide. In particularexamples, the metal ion is a cold metal ion (not a radionuclide). In aparticular embodiments, the cold metal ion is Re-187. In other examples,the metal ion is a radionuclide. Examples of metal ions include any ofthose metal ions discussed above. In some embodiments, the kit includesone or more vials containing a composition comprising disodium hydrogenphosphate dehydrate, mannitol, ascorbic acid, sodium edentate, stannouschloride dehydrate, tartaric acid, or potassium dihydrogen-phosphate,and a pharmaceutically acceptable carrier.

Further embodiments of the present invention pertain to an imaging,therapeutic, or radio/therapeutic agent, prepared by a method comprisingany of the methods set forth above. In some embodiments, thechelator-targeting ligand conjugate is between about 90% and about 99.9%pure. In some embodiments, the chelator-targeting ligand conjugate isbetween about 80% and about 99.9% pure. In some embodiments, thechelator-targeting ligand conjugate is between about 70% and about 99.9%pure. In specific embodiments, the metal ion-labeled chelator-targetingligand conjugate comprises ethylenedicysteine. In particularembodiments, the metal ion labeled chelator-targeting ligand conjugateis ^(99m)Tc-EC-glucosamine. In further particular embodiments, the metalion labeled chelator-targeting ligand conjugate is ¹⁸⁶Re-EC-glucosamine.In still further embodiments, the metal ion labeled chelator-targetingligand conjugate is ¹⁸⁷Re-EC-glucosamine.

Further embodiments pertain to a method of imaging, diagnosing, ortreating a subject, comprising administering to the subject apharmaceutically or diagnostically effective amount of a metal ionlabeled chelator-targeting ligand conjugate, wherein thechelator-targeting ligand conjugate is prepared by a method comprisingany of the methods set forth above, wherein the disease is imaged,diagnosed, or treated. In certain embodiments, the metal ionlabeled-chelator conjugate is between about 90% and about 99.9% pure. Incertain embodiments, the metal ion labeled-chelator conjugate is betweenabout 80% and about 99.9% pure. In certain embodiments, the metal ionlabeled-chelator conjugate is between about 70% and about 99.9% pure. Inparticular embodiments, the metal ion labeled-chelator conjugatecomprises ethylenedicysteine. The metal ion, for example, may be any ofthose metal ions set forth above.

Certain embodiments pertain to a method of treating a subject with ahyperproliferative disease, comprising administering to the subject apharmaceutically effective amount of a metal ion-labeledchelator-targeting ligand conjugate prepared by any of the methods setforth herein. In particular embodiments, the hyperproliferative diseaseis cancer. For example, the cancer may be breast cancer, lung cancer,prostate cancer, ovarian cancer, brain cancer, liver cancer, cervicalcancer, colon cancer, renal cancer, skin cancer, head and neck cancer,bone cancer, a esophageal cancer, bladder cancer, uterine cancer,lymphatic cancer, stomach cancer, pancreatic cancer, testicular cancer,lymphoma, or leukemia. In certain embodiments, the method is furtherdefined as a method for performing dual radio/chemotherapy. Someembodiments further comprise administering one or more secondary formsof therapy of a hyperproliferative disease. For example, the secondaryform of therapy may be chemotherapy, gene therapy, surgical therapy,radiation therapy, or immunotherapy. Certain embodiments pertain tomethods of performing dual imaging and therapy in a subject.

Embodiments of the present invention also generally pertain to methodsof diagnosis, assessing efficacy of treatment, or imaging in a subjectwith known or suspected cardiovascular disease. The subject can be anysubject, such as a mammal or animal models used to assess the presenceof cardiovascular disease. The mammal, for example, may be a human ormember of the monkey species. Animal models include dogs, cats, rats,mice or rabbits. In preferred embodiments, the subject is a human withknown or suspected cardiovascular disease.

The cardiovascular disease can be any disease of the heart or tissuenourished by the vascular system. The vascular system includes coronaryarteries, and all peripheral arteries supplying nourishment to theperipheral vascular system and the brain. The vascular system includesarteries, veins, arterioles, venules, and capillaries. Examples ofcardiovascular diseases include diseases of the heart, such asmyocardial infarction, myocardial ischemia, angina pectoris, congestiveheart failure, cardiomyopathy (congenital or acquired), arrhythmia, orvalvular heart disease. In particular embodiments, the subject is knownor suspected to have myocardial ischemia.

The subject, for example, may be a patient who presents to a clinic withsigns or symptoms suggestive of myocardial ischemia or myocardialinfarction. Imaging of the heart of the subject to diagnose disease mayinvolve administering to the subject a pharmaceutically effective amountof a metal ion labeled chelator-targeting ligand conjugate synthesizedusing any of the methods set forth herein. Imaging can be performedusing any imaging modality known to those of ordinary skill in the art.In particular embodiments, imaging involves use radionuclide-basedimaging technology, such as PET or SPECT. In particular embodiments, themetal ion-labeled radionuclide-targeting ligand conjugate is99m-Tc-EC-glucosamine. Glucosamine is not actively taken up by viablemyocardial tissue but rather is target specific for regions of ischemia.Severity of ischemia can be visually assessed or graded depending onmagnitude of the signal that is measured using any method known to thoseof ordinary skill in the art. In some embodiments, imaging using any ofthe conjugates set forth herein is performed before, during, or afterimaging of the heart using a second imaging agent. For example, thesecond imaging agent may be thallium imaged by scintigraphy to woulddefine the region of normal myocardial perfusion (non-ischemic tissue).

Myocardial perfusion SPECT (MPS) consist of a combination of a stressmodality (exercise or pharmacologic) with rest and stress administrationand imaging of radiopharmaceuticals. Thallium has excellent physiologicproperties for myocardial perfusion imaging. Being highly extractedduring the first pass through the coronary circulation, a linearrelationship between blood flow to viable myocardium and thallium uptakehas been shown during exercise; however, at very high levels of flow, a“roll-off” in uptake occurs. As an unbound potassium analogue, thalliumredistributes over time. Its initial distribution is proportional toregional myocardial perfusion and at equilibrium, the distribution ofthallium is proportional to the regional potassium pool, reflectingviable myocardium. The mechanisms of thallium redistribution aredifferential washout rates between hypoperfused but viable myocardiumand normal zones and wash-in to initially hypoperfused zones. Thewashout rate of thallium is the concentration gradient between themyocardial cell and the blood. There is slower blood clearance ofthallium following resting or low-level exercise injection. Diffuse slowwashout rates, mimicking diffuse ischemia, may be observed in normalpatients who do not achieve adequate levels of stress. Hyperinsulinemicstates slow redistribution, leading to an underestimation of viablemyocardium; thus fasting is recommended prior to and for 4 hrs followingthallium injection. This is why if EC-G is used as an viable agent incombination with thallium it will target the precise area of interestwhich would be the Ischemic but viable area (see Angello et al., 1987;Gutman et al., 1983; Pohost et al., 1977).

Imaging using any of the metal ion-labeled chelator-targeting ligandconjugates of the present invention may also be performed in conjunctionwith other diagnostic methods, such as measurement of cardiac isozymes,or cardiac catheterization. The imaging may be performed at variousintervals following onset of symptoms, or can be performed to assess forchanges in myocardial perfusion over time.

Further embodiments pertain to a method of imaging a site within asubject comprising (a) administering to the subject a diagnosticallyeffective amount of a metal ion labeled-chelator-targeting ligandconjugate, wherein the metal ion-labeled chelator-targeting ligandconjugate is synthesized by any of the methods set forth herein; and (b)detecting a signal from the metal ion labeled-chelator-targeting ligandconjugate that is localized at the site. In certain embodiments, themetal ion labeled-chelator-targeting ligand conjugate is between about90% and about 99.9% pure. In specific embodiments, the metal ionlabeled-chelator-targeting ligand conjugate comprisesethylenedicysteine.

The signal can be detected by any method known to those of ordinaryskill in the art. Non-limiting examples of such methods include PET,PET/CT, CT, SPECT, SPECT/CT, MRI, optical imaging and ultrasound.

The subject can be any subject, such as a mammal or avian species. Inparticular embodiments, the mammal is a human. The site to be imaged canbe any site in a subject, and may include, for example, a tumor, heart,lung, esophagus, muscle, intestine, breast, prostate, stomach, bladder,liver, spleen, pancreas, kidney, a tumor, duodenum, jejunum, ileum,cecum, colon, rectum, salivary gland, gall bladder, urinary bladder,trachea, larynx, pharynx, aorta, artery, vein, thymus, lymph node, bone,pituitary gland, thyroid gland, parathyroid gland, adrenal gland, brain,cerebrum, cerebellum, medulla, pons, spinal cord, nerve, skeletalmuscle, smooth muscle, bone, testes, epidiymides, prostate, seminalvesicles, penis, ovary, uterus, mammary gland, vagina, skin, eyes, oroptic nerve. In particular embodiments, the site to be imaged is atumor. In further particular embodiments, the site to be imaged is theheart.

In some embodiments, the method of imaging further comprises performingone or more additional diagnostic or imaging procedures to evaluated thesubject for a disease. In further embodiments, the method of imaging isfurther defined as a method of performing dual imaging and therapy.

In certain embodiments, the disease to be treated is a cardiovasculardisease. Non-limiting examples of such diseases include myocardialinfarction, congestive heart failure, cardiomyopathy, valvular heartdisease, an arrhythmia, congenital heart disease, and angina pectoris.

The present invention also generally pertains to methods for imaging thebrain or spinal cord (neuroendocrine system) of a subject, comprisingadministering to a subject one or more of the conjugates of the presentinvention. In some embodiments, for example, the chelate is conjugatedto a targeting ligand that is capable of crossing the blood-brainbarrier of a subject. A non-limiting example of such a targeting ligandis an amino acid, such as tyrosine or an analog of tyrosine such asalpha-methyl tyrosine. Other examples include somatostatin, octreotide,and tryptophan.

The present invention also generally pertains methods of treating asubject with a disorder of the central nervous system of a subject. Thedisorder of the central nervous system may be, for example, aneurodegenerative disease such as Parkinson's disease, Huntington'sdisease, amyotrophic lateral sclerosis, Alzheimer disease, or aneuroendocrine tumor. Examples of neuroendocrine tumors include primaryand metastatic brain tumors. Examples of primary brain tumors includeastrocytomas, glioblastomas, oligodendrogliomas, ependymomas, mixedgliomas, mixed glio-neuronal tumors (tumors displaying a neuronal, aswell as a glial component, e.g. gangliogliomas, disembryoplasticneuroepithelial tumors) and tumors originating from neuronal cells(e.g., gangliocytoma, central gangliocytoma). The tumor may be ametastatic tumor. In some embodiments, the disorder of the centralnervous system is an inflammatory disease. For example, the disease maybe an infectious disease, or an immune disease.

The present invention also pertains to a method of determining thepurity of a composition comprising a metal ionlabeled-chelator-targeting ligand conjugate of unknown purity is alsocontemplated by the present invention, said method comprising:

-   -   a) obtaining a first composition comprising a metal ion        labeled-chelator-targeting ligand conjugate of unknown purity;    -   b) obtaining a second composition comprising a metal ion        labeled-chelator-targeting ligand conjugate prepared by any of        the methods described herein;    -   c) performing quantitative analysis on a sample of the first        composition to generate a first measurement;    -   d) performing quantitative analysis of the second composition to        generate a second measurement; and    -   e) calculating a ratio of the first measurement to the second        measurement, wherein the ratio of the first measurement to the        second measurement is a measure of purity of the composition        comprising a metal ion labeled-chelator-targeting ligand        conjugate of unknown purity.

Quantitative analysis may be performed via any technique known to thoseof skill in the art. In certain embodiments, quantitative analysis isperformed by technique selected from the group consisting ofautoradiography, dialysis, mass spectroscopy, melting pointdetermination, ultra violet analysis, colorimetric analysis,high-performance liquid chromatography, thin-layer chromatography andnuclear magnetic resonance analysis.

Other aspects of the present invention contemplate a compositioncomprising a chelator-targeting ligand conjugate, wherein the chelatoris of the following formula:

wherein:

-   -   the point of conjugation between the chelator and the targeting        ligand is at one or more positions selected from the group        consisting of A, B, C, D, E and F;    -   A, D, E and F are each independently H, lower alkyl, —COOH,        —NH₂, or thiol, with the proviso that at least one position is        —NH₂ or thiol;    -   B and C are each independently a secondary amine, a tertiary        amine, —S—, —S(O)—, or —S(O)₂—;    -   R₁, R₂, R₃ and R₄ are each independently H or lower alkyl; and    -   X is selected from the group consisting of —CH₂—CH₂—,        —CH₂—CH₂—CH₂—, —CH₂—C(O)—, —C(O)—CH₂—, —C(O)—CH₂—CH₂— and        —CH₂—CH₂—C(O)—;        wherein at least one of A, B, C, D, E, F, or one functional        group of the targeting ligand is protected by a protecting        group, and        wherein the chelator-targeting ligand conjugate is between about        75% and about 99.9% pure.

The protecting group may be of any type described herein. The targetingligand may be of any type described herein. In certain embodiments, thecomposition has the proviso that when A and D are each —NH₂, neither Bnor C is a secondary or a tertiary amine. The composition may comprise achelator-targeting ligand conjugate that is between about 70% and about99.9% pure. The composition may comprise a chelator-targeting ligandconjugate that is between about 80% and about 99.9% pure. Thecomposition may comprise a chelator-targeting ligand conjugate that isbetween about 85% and about 99.9% pure. The composition may comprise achelator-targeting ligand conjugate that is between about 90% and about99.9% pure. The composition may comprise a chelator-targeting ligandconjugate that is between about 95% and about 99.9% pure. Thecomposition may, in certain embodiments, be further defined as a metalion labeled-chelator-targeting ligand conjugate, as discussed herein.

Another aspect of the present invention contemplates a compositioncomprising a chelator-targeting ligand conjugate, wherein the chelatoris of the following formula:

wherein:

-   -   the point of conjugation between the chelator and the targeting        ligand is at one or more positions selected from the group        consisting of A, B, C, D, E and F;    -   A, D, E and F are each independently H, lower alkyl, —COOH,        —NH₂, or thiol, with the proviso that at least one position is        —NH₂ or thiol;    -   B and C are each independently a secondary amine, a tertiary        amine, —S—, —S(O)—, or —S(O)₂—;    -   R₁, R₂, R₃ and R₄ are each independently H or lower alkyl; and    -   X is selected from the group consisting of —CH₂—CH₂—,        —CH₂—CH₂—CH₂—, —CH₂—C(O)—, —C(O)—CH₂—, —C(O)—CH₂—CH₂— and        —CH₂—CH₂—C(O)—;        wherein the chelator-targeting ligand conjugate is between about        75% and about 99.9% pure.

The targeting ligand may be of any type described herein. In certainembodiments, the composition has the proviso that when A and D are each—NH₂, neither B nor C is a secondary or a tertiary amine. Thecomposition may comprise a chelator-targeting ligand conjugate that isbetween about 70% and about 99.9% pure. The composition may comprise achelator-targeting ligand conjugate that is between about 80% and about99.9% pure. The composition may comprise a chelator-targeting ligandconjugate that is between about 85% and about 99.9% pure. Thecomposition may comprise a chelator-targeting ligand conjugate that isbetween about 90% and about 99.9% pure. The composition may comprise achelator-targeting ligand conjugate that is between about 95% and about99.9% pure. The composition may, in certain embodiments, be furtherdefined as a metal ion labeled-chelator-targeting ligand conjugate, asdiscussed herein. The composition may be further defined as^(99m)Tc-EC-glucosamine. The composition may be further defined as¹⁸⁶Re-EC-glucosamine. The composition may be further defined as¹⁸⁷Re-EC-glucosamine.

It is contemplated that any embodiment discussed in this specificationcan be implemented with respect to any method, compound or compositionof the invention, and vice versa. Furthermore, compounds andcompositions of the invention can be used to achieve methods of theinvention.

A person of ordinary skill in the art will recognize that chemicalmodifications can be made to the compounds of the present invention, aswell as compounds employed in the method of the present invention,without departing from the spirit and scope of the present invention.Substitutes, derivatives, or equivalents can also be used, all of whichare contemplated as being part of the present invention.

As used herein, “organic medium” refers to solutions (e.g., reactionsolutions) and purification methods comprising one or more organicsolvents (also called “solvents” herein). Solvent choices for themethods of the present invention will be known to one of ordinary skillin the art. Solvent choices may depend, for example, on which one(s)will facilitate the solubilizing of all the reagents, or, for example,which one(s) will best facilitate the desired reaction (particularly ifthe mechanism of the reaction is known). Solvents may include, forexample, polar solvents and non-polar solvents. Solvents choicesinclude, but are not limited to, dimethylformamide, dimethylsulfoxide,dioxane, methanol, ethanol, hexane, methylene chloride and acetonitrile.In some preferred embodiments, solvents include ethanol,dimethylformamide and dioxane. More than one solvent may be chosen forany particular reaction or purification procedure. Water (i.e., anaqueous component) may also be admixed into any solvent choice; water istypically added to facilitate solubilization of all the reagents. Incertain embodiments, the organic component of the organic medium, byvolume, is about or at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% organic solventcompared to the aqueous component.

The word “conjugate” and “conjugated” is defined herein as chemicallyjoining within the same molecule. For example, two or more moleculesand/or atoms may be conjugated together via a covalent bond, forming asingle molecule. The two molecules may be conjugated to each other via adirect connection (e.g., where the compounds are directly attached via acovalent bond) or the compounds may be conjugated via an indirectconnection (e.g., where the two compounds are covalently bonded to oneor more linkers, forming a single molecule). In other instances, a metalatom may be conjugated to a molecule via a chelation interaction.

The term “functional group” generally refers to how persons of skill inthe art classify chemically reactive groups. Non-limiting examples offunctional groups include carbon-carbon bonds (including single, doubleand triple bonds), hydroxyl (or alcohol), amine, sulfhydryl (or thiol),amide, ether, ester, thioether, thioester, carboxylic acid and carbonylgroups. As used herein, “amine” and “amino” and other similar pairs ofwords such as “hydroxy” and “hydroxyl” refer to the same functionalmoiety and thus are used interchangeably. As used herein, “amine” mayrefer to either or both —NH₂ and —NH—.

As used herein, “chelate” may be used as a noun or a verb. As a noun,“chelate” refers to one or more atoms that are either capable ofchelating one or more metal ions, or are chelating to one or more metalions. In preferred embodiments, only one metal ion coordinates to achelate. A non-limiting example of “chelate” includes “an N₂S₂” chelate:this means that two nitrogen atoms and two sulfur atoms of a chelatorare either a) capable of chelating to one or more metal ions or b) arecoordinated to (or chelated to) to one or more metal ions (preferablyjust one metal ion). As a verb, “chelate” refers to the process of ametal ion becoming coordinated or chelated to, for example, a chelatoror a chelator-targeting ligand conjugate.

As used herein, an “unconjugated chelator” refers to a chelator that isnot conjugated to a targeting ligand.

As used herein, an “unprotected chelator” refers to a chelator that doesnot comprise any protecting groups.

As used herein, a “protected chelator” refers to a chelator thatcomprises at least one protecting group.

As used herein, an “unprotected targeting ligand” refers to a targetingligand that does not comprise any protecting groups.

As used herein, a “protected targeting ligand” refers to a targetingligand that comprises at least one protecting group.

The term “nucleophile” or “nucleophilic” generally refers to atomsbearing one or more lone pairs of electrons. Such terms are well knownin the art and include —NH₂, thiolate, carbanion and alcoholate (alsoknown as hydroxyl).

The term “electrophile” or “electrophilic” generally refers to speciesthat react with nucleophiles. Electrophilic groups typically have apartial positive charge. Such a term is well known in the art andincludes the carbon of a carbon bonded to a leaving group such as ahalogen, sulfonyl, or a quaternary amino group.

The term “leaving group” generally refers to groups readily displaceableby a nucleophile, such as an amine, and alcohol or a thiol nucleophile.Such leaving groups are well known and include carboxylates,N-hydroxysuccinimide, N-hydroxybenzotriazole, halogen (halides),triflates, tosylates, mesylates, alkoxy, thioalkoxy, sulfonyls and thelike.

As used herein, “alkyl” or “alk” refers to a straight, branched orcyclic carbon-carbon or hydrocarbon chain, optionally including alkeneor alkyne bonding, containing 1-30 carbons. “Lower alkyl” refers toalkyl radicals comprising 1-4 carbons. Non-limiting examples of loweralkyls include methyl, ethyl, propyl, butyl and isopropyl. “Substitutedalkyl” refers to an alkyl radical substituted with at least one atomknown to those of skill in the art. In certain embodiments, one or moresubstituents may be selected from the group consisting of hydrogen,halogen, oxo (e.g., ether), hydroxy, alkoxy, silyloxy, cycloalkyl, acyl,aryl, acetyl, carbonyl, thiocarbonyl, cyano, azido, amido,aminocarbonyl, amino, —NH-alkyl, —N(alkyl)₂, —NH-cycloalkyl,—N(cycloalkyl)₂, —NH-aryl, —N(aryl)₂, trialkylsilyloxy, acyloxy,acylamino, bis-acylamino, ester, NO, NO₂ and sulfo (e.g., thioether,thioester, sulfonamido, sulfonyl).

The term “aryl” refers to a carbocyclic aromatic group, including butnot limited to those selected from the group consisting of phenyl,naphthyl, indenyl, indanyl, azulenyl, fluorenyl, and anthracenyl; or aheterocyclic aromatic group, including but not limited to those selectedfrom the group consisting of furyl, furanyl, thienyl, pyridyl, pyrrolyl,oxazolyl, thiazolyl, imidazolyl, pyrazolyl, pyrazolinyl, pyrazolidinyl,isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiadiazolyl,pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, trithianyl, indolizinyl,indolyl, isoindolyl, indolinyl, thiophenyl, indazolyl, benzimidazolyl,benzthiazolyl, purinyl, quinolizinyl, quinolinyl, isoquinolinyl,innolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, naphthyridinyl,pteridinyl carbazolyl, acridinyl, phenazinyl, phenothiazinyl,phenoxazinyl and any combination or derivative of one or more of thesegroups.

“Aryl” groups, as defined in this application may independently containone or more functional groups as substituents. In certain embodiments,substituents may be selected from the group consisting of hydrogen,alkyl, halogen, oxo (e.g., ether), hydroxy, alkoxy, silyloxy,cycloalkyl, acyl, aryl, acetyl, carbonyl, thiocarbonyl, cyano, amido,aminocarbonyl, amino, —NH-alkyl, —N(alkyl)₂, —NH-cycloalkyl,—N(cycloalkyl)₂, —NH-aryl, —N(aryl)₂, trialkylsilyloxy, acyloxy,acylamino, bis-acylamino, ester, NO, NO₂ and sulfo (e.g., thioether,thioester, sulfonamido, sulfonyl). Further, any of these substituentsmay be further substituted with substituents as just described.

As used herein the term “cycloalkyl” refers to carbocycles orheterocycles of three or more atoms, the ring atoms of which may beoptionally substituted with C, S, O or N, and the ring atoms of whichmay comprise one or more functional group as substituents. Substituentsmay be selected, in some embodiments, from the group consisting ofhydrogen, alkyl, halogen, oxo (e.g., ether), hydroxy, alkoxy, silyloxy,cycloalkyl, acyl, aryl, acetyl, carbonyl, thiocarbonyl, cyano, azido,amido, aminocarbonyl, amino, —NH-alkyl, —N(alkyl)₂, —NH-cycloalkyl,—N(cycloalkyl)₂, —NH-aryl, —N(aryl)₂, trialkylsilyloxy, acyloxy,acylamino, bis-acylamino, ester, NO, NO₂ and sulfo (e.g., thioether,thioester, sulfonamido, sulfonyl).

The term “amino acid” refers to any of the naturally occurring aminoacids, as well as synthetic analogs (e.g., D-stereoisomers of thenaturally occurring amino acids, such as D-threonine) and derivativesthereof. α-Amino acids comprise a carbon atom to which is bonded anamino group, a carboxyl group, a hydrogen atom, and a distinctive groupreferred to as a “side chain.” Amino acids comprising an additionalmethylene group in their backbone are often called β-amino acids. Theside chains of naturally occurring amino acids are well known in the artand include, for example, hydrogen (e.g., as in glycine), alkyl (e.g.,as in alanine, valine, leucine, isoleucine, proline), substituted alkyl(e.g., as in threonine, serine, methionine, cysteine, aspartic acid,asparagine, glutamic acid, glutamine, arginine, and lysine), arylalkyl(e.g., as in phenylalanine and tryptophan), substituted arylalkyl (e.g.,as in tyrosine), and heteroarylalkyl (e.g., as in histidine). Unnaturalamino acids are also known in the art, as set forth in, for example,Williams (1989); Evans et al. (1990); Pu et al. (1991); Williams et al(1991); and all references cited therein. The present invention includesthe side chains of unnatural amino acids as well.

The terms “primary amine,” “secondary amine” and “tertiary amine” referto amines, as derivatives of ammonia (NH₃), in which one (primary), two(secondary) or three (tertiary) of the hydrogens have been replaced bycarbon, wherein said carbon may be attached to any other atom. Incertain embodiments, said carbon (C) is comprised in X of the formulashown above, a hydrocarbon group (e.g., —CH₂—), —CH(E)(CHAR₁R₂),—CH(F)(CHDR₃R₄), or a —C(O)— group, wherein A, D, E, F, X, R₁, R₂, R₃and R₄ are as defined herein.

Compounds as described herein may contain one or more asymmetric centersand thus can occur as racemates and racemic mixtures, singleenantiomers, diastereomeric mixtures and individual diasteromers. Allpossible stereoisomers of the all the compounds described herein, unlessotherwise noted, are contemplated as being within the scope of thepresent invention. The chiral centers of the compounds of the presentinvention can have the S- or the R-configuration, as defined by theIUPAC 1974 Recommendations. The present invention is meant to comprehendall such isomeric forms of the compounds of the invention.

The claimed invention is also intended to encompass salts of any of thesynthesized compounds of the present invention. The term “salt(s)” asused herein, is understood as being acidic and/or basic salts formedwith inorganic and/or organic acids and bases. Zwitterions (internal orinner salts) are understood as being included within the term “salt(s)”as used herein, as are quaternary ammonium salts such as alkylammoniumsalts. Nontoxic, pharmaceutically acceptable salts are preferred asdescribed below, although other salts may be useful, as for example inisolation or purification steps.

Non-limiting examples of acid addition salts include but are not limitedto acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate,bisulfate, butyrate, citrate, camphorate, camphorsulfonate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate,hexanoate, hydrochloride, hydrobromide, hydroiodide,2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate,2-naphthalenesulfonate, nicotinate, oxalate, pectinate, persulfate,3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate,thiocyanate, tosylate and undecanoate.

Non-limiting examples of basic salts include but are not limited toammonium salts; alkali metal salts such as sodium, lithium, andpotassium salts; alkaline earth metal salts such as calcium andmagnesium salts; salts comprising organic bases such as amines (e.g.,dicyclohexylamine, alkylamines such as t-butylamine and t-amylamine,substituted alkylamines, aryl-alkylamines such as benzylamine,dialkylamines, substituted dialkylamines such as N-methyl glucamine(especially N-methyl D-glucamine), trialkylamines, and substitutedtrialkylamines); and salts comprising amino acids such as arginine,lysine and so forth. The basic nitrogen-containing groups may bequaternized with agents such as lower alkyl halides (e.g. methyl, ethyl,propyl, and butyl chlorides, bromides and iodides), dialkyl sulfates(e.g. dimethyl, diethyl, dibutyl, and diamyl sulfates), long chainhalides (e.g. decyl, lauryl, myristyl and stearyl chlorides, bromidesand iodides), arylalkyl halides (e.g. benzyl and phenethyl bromides) andothers known in the art.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.”

Throughout this application, the term “about” is used to indicate that avalue includes the inherent variation of error for the device, themethod being employed to determine the value, or the variation thatexists among the study subjects. For example, “about” can be within 10%,preferably within 5%, more preferably within 1%, and most preferablywithin 0.5%.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.”

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1. Non-limiting example of an organic synthesis ofethylenedicysteine-glucosamine (EC-G).

FIG. 2. Non-limiting example of an organic synthesis ofrhenium-ethylenedicysteine-glucosamine (Re-EC-G).

FIG. 3. [³H]Thymidine incorporation assay using Re-EC-G and a lymphomacell line.

FIG. 4. Comparison of cellular uptake of Ec-G in crude form or prepHPLC-purified form.

FIG. 5. Mass spectrometry of EC-G.

FIG. 6. Radio-TLC (thin layer chromatography) of ⁶⁸Ga-EC-G. (a)⁶⁸Ga-EC-G, synthesized via organic means; (b) ⁶⁸Ga-EC-G, synthesized viaaqueous means; (c) free ⁶⁸Ga.

FIG. 7. Analytic radio-HPLC of ⁶⁸Ga-EC-G. (a) UV detection; (b) NaIdetection.

FIGS. 8A and 8B. Stability of ⁶⁸Ga-EC-G in dog serum as shown byradio-TLC. (a) ⁶⁸Ga-EC-G (0.7 mg/0.7 ml, pH 7.5, 865 μCi); (b) 100 μL⁶⁸Ga-EC-G in 100 μL dog serum, time=0; (c) time=30 min.; (d) time=60min.; (e) time=120 min.; (f) ⁶⁸Ga-EC-BSA.

FIG. 9. Stability of ⁶⁸Ga-EC-G in dog serum as analyzed in a proteinbinding assay.

FIG. 10. In vitro uptake study of ⁶⁸Ga-labeled compounds in breastcancer cell line 13762.

FIG. 11. Planar images of the ^(99m)Tc-EC-ESMOLOL derivative (300μCi/rat) in breast tumor-bearing rats. H/UM=heart/upper mediastinumcount density (counts/pixel) ratios at 15-45 minutes.

FIG. 12. ⁶⁸Ga-EC-TML PET imaging in a New Zealand white rabbit.

FIG. 13. Non-limiting example of an organic synthesis ofethylenedicysteine-glucosamine (EC-G).

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present inventors have identified novel synthetic methods for thepreparation of chelator-targeting ligand conjugates optionally chelatedto one or more metal ions. The present invention further providessyntheses of chelators, such as unconjugated chelators, protectedchelators (that is, chelators wherein one or more functional groups areprotected using a protecting agent) and metal ion labeled-chelators(that is, chelators that are chelated to one or more metal ions). Thesesynthetic methods comprise, generally, the use of organic solvents andsynthetic organic procedures and purification methods. Methods based onwet (aqueous) chemistry are also provided. Compounds of the presentinvention resulting from such organic chemistry methods are high inpurity, especially when compared to compounds prepared by wet chemistry.A preferred chelator is ethylenedicysteine. The targeting ligand can be,for example, a tissue-targeting moiety, a diagnostic moiety, or atherapeutic moiety. The metal ions chelated to compounds of the presentinvention may further render the compound useful for imaging,diagnostic, or therapeutic use. Compounds of the present invention,methods of their synthesis and use are further described below.

A. Chelators

Persons of skill in the art will be familiar with compounds capable ofchelating one or more metal ions (“chelators”). Chelators employed inthe method of the present invention generally comprise one or more atomscapable of chelating to one or more metal ions. Chelators comprisingthree or four atoms available for chelation are preferred. Typically, achelator chelates to one metal ion.

Chelation of a metal ion to a chelator can be by any method known tothose of ordinary skill in the art. Methods of chelation (also calledcoordination) are described in more detail below. Atoms available forchelation are known to those of skill in the art, and typically compriseO, N or S. In preferred embodiments, the atoms available for chelationare selected from the group consisting of N and S. In certain preferredembodiments, the metal ion is chelated to a group of atoms, referred toherein as “chelates,” selected from the group consisting of NS₂, N₂S,S₄, N₂S₂, N₃S and NS₃. Chelation can also occur among both the chelatorand the targeting ligand—i.e., both the chelator and the targetingligand may contribute atoms that chelate the same metal ion.

In certain embodiments, the chelator comprises compounds incorporatingone or more amino acids. Amino acids will typically be selected from thegroup consisting of cysteine and glycine. For example, the chelator maycomprise three cysteines and one glycine or three glycines and onecysteine. As discussed below, a spacer may connect one amino acid toanother.

It is well known to those of ordinary skill in the art that chelators,in general, comprise a variety of functional groups. Non-limitingexamples of such functional groups include hydroxy, thiol, amine, amidoand carboxylic acid.

1. Bis-Aminoethanethiol (BAT) Dicarboxylic Acids

Bis-aminoethanethiol (BAT) dicarboxylic acids may constitute a chelatoremployed in the method of the present invention. In preferredembodiments, the BAT dicarboxylic acid is ethylenedicysteine (EC). BATdicarboxylic acids are capable of acting as tetradentate ligands, andare also known as diaminodithiol (DADT) compounds. Such compounds areknown to form stable Tc(V)O-complexes on the basis of efficient bindingof the oxotechnetium group to two thiol-sulfur and two amine-nitrogenatoms. The ^(99m)Tc labeled diethylester (^(99m)Tc-L,L-ECD) is known asa brain agent. ^(99m)Tc-L,L-ethylenedicysteine (^(99m)Tc-L,L-EC) is itsmost polar metabolite and was discovered to be excreted rapidly andefficiently in the urine. Thus, ^(99m)Tc-L,L-EC has been used as a renalfunction agent. (Verbruggen et al. 1992). Other metals such as indium,rhenium, gallium, copper, holmium, platinum, gadolinium, lutetium,yttrium, cobalt, calcium and arsenic may also be chelated to BATdicarboxylic acids such as EC.

2. Spacers

Chelators of the present invention may comprise one or more spacers. Forexample, amino acids and their derivatives may be joined by one or morespacers. An example of two amino acids joined by a spacer includesethylenedicysteine, described above. Such spacers are well known tothose of ordinary skill in the art. These spacers, in general, provideadditional flexibility to the overall compound that may facilitatechelation of one or more metal ions to the chelator. Non-limitingexamples of spacers include alkyl groups of any length, such as ethylene(—CH₂—CH₂—), ether linkages, thioether linkages, amine linkages and anycombination of one or more of these groups. It is envisioned thatmultiple chelators (that is, two or more) linked together are capable offorming an overall molecule that may chelate to one or more, or moretypically two or more, metal ions. That is, each chelator that makes upthe overall molecule may each chelate to a single separate metal ion.

B. Protecting Groups

When a chemical reaction is to be carried out selectively at onereactive site in a multifunctional compound, other reactive sites oftenmust be temporarily blocked. A “protecting group,” as used herein, isdefined as a group used for the purpose of this temporary blockage.Thus, the function of a protecting group is to protect one or morefunctional groups (e.g., —NH₂, —SH, —COOH) during subsequent reactionswhich would not proceed well, either because the free (in other words,unprotected) functional group would react and be functionalized in a waythat is inconsistent with its need to be free for subsequent reactions,or the free functional group would interfere in the reaction. Persons ofskill in the art recognize that the use of protecting groups is typicalin synthetic organic chemistry.

During the synthesis of the compounds of the present invention, variousfunctional groups must be protected using protecting agents at variousstages of the synthesis. A “protecting agent” is used to install theprotecting group. Thus, in a typical procedure, a protecting agent isadmixed with a compound featuring a functional group that is to beprotected, and the protecting agent forms a covalent bond with thatfunctional group. In this manner, the functional group is “protected” bya protecting group (and effectively rendered unreactive) by the covalentbond that formed with the protecting agent. Multiple functional groupscan be protected in one or more steps using properly selected protectingagents. Such proper selection is understood by those of skill in theart. Such selection is often based upon the varying reactivity of thefunctional groups to be protected: thus, more reactive groups (such assulfur/thiol) are typically protected before less reactive groups (suchas amine) are protected.

There are a number of methods well known to those skilled in the art foraccomplishing such a step. For protecting agents, their reactivity,installation and use, see, e.g., Greene and Wuts (1999), hereinincorporated by reference in its entirety. The same protecting group maybe used to protect one or more of the same or different functionalgroup(s). Non-limiting examples of protecting group installation aredescribed below.

Use of the phrase “protected hydroxy” or “protected amine” and the likedoes not mean that every such functional group available to be protectedis protected. Similarly, a “protected chelator,” as used herein, doesnot imply that every functional group of the chelator is protected.

Compounds of the present invention, including compounds used and madeduring the practice of the method of the present invention, arecontemplated both in protected and unprotected (or “free”) form. Personsof ordinary skill in the art will understand that functional groupsnecessary for a desired transformation should be unprotected.

When a protecting group is no longer needed, it is removed by methodswell known to those skilled in the art. For deprotecting agents andtheir use, see, e.g., Greene and Wuts (1999). Agents used to remove theprotecting group are typically called deprotecting agents. Protectinggroups are typically readily removable (as is known to those skilled inthe art) by methods employing deprotecting agents that are well known tothose skilled in the art. For instance, acetate ester and carbamateprotecting groups may be easily removed using mild acidic or basicconditions, yet benzyl and benzoyl ester protecting groups need muchstronger acidic or basic conditions. It is well known that certaindeprotecting agents remove some protective groups and not others, whileother deprotecting agents remove several types of protecting groups fromseveral types of functional groups. For instance, Birch reductionreactions using liquid ammonia and sodium (as described below) deprotectbenzyl groups from thiols (or sulfur, more particularly) or carbamategroups from nitrogen, but not acetate groups from oxygen. Thus, a firstdeprotecting agent may be used to remove one type of protecting group,followed by the use of a second deprotecting agent to remove a secondtype of protecting group, and so on.

Persons of ordinary skill in the art will be familiar with the properordering of protective group removal using deprotecting agents. Seee.g., Greene and Wuts (1999). Non-limiting examples of protecting groupremoval are discussed below.

Amine protecting groups are well known to those skilled in the art. See,for example, Greene and Wuts (1999), Chapter 7. These protecting groupscan be installed via protecting agents well known to those of skill inthe art. Removal of these groups is also well known to those of skill inthe art.

In some embodiments, the amine protecting group may be selected from thegroup consisting of t-butoxycarbonyl, benzyloxycarbonyl, formyl, trityl,acetyl, trichloroacetyl, dichloroacetyl, chloroacetyl, trifluoroacetyl,difluoroacetyl, fluoroacetyl, benzyl chloroformate,4-phenylbenzyloxycarbonyl, 2-methylbenzyloxycarbonyl,4-ethoxybenzyloxycarbonyl, 4-fluorobenzyloxycarbonyl,4-chlorobenzyloxycarbonyl, 3-chlorobenzyloxycarbonyl,2-chlorobenzyloxycarbonyl, 2,4-dichlorobenzyloxycarbonyl,4-bromobenzyloxycarbonyl, 3-bromobenzyloxycarbonyl,4-nitrobenzyloxycarbonyl, 4-cyanobenzyloxycarbonyl,2-(4-xenyl)isopropoxycarbonyl, 1,1-diphenyleth-1-yloxycarbonyl,1,1-diphenylprop-1-yloxycarbonyl, 2-phenylprop-2-yloxycarbonyl,2-(p-toluoyl)prop-2-yloxycarbonyl, cyclopentanyloxycarbonyl,1-methylcyclopentanyloxycarbonyl, cyclohexanyloxycarbonyl,1-methylcyclohexanyloxycabonyl, 2-methylcyclohexanyloxycarbonyl,2-(4-toluylsulfonyl)ethoxycarbonyl, 2-(methylsulfonyl)ethoxycarbonyl,2-(triphenylphosphino)ethoxycarbonyl, fluorenylmethoxycarbonyl,2-(trimethylsilyl)ethoxycarbonyl, allyloxycarbonyl,1-(trimethylsilylmethyl)prop-1-enyloxycarbonyl,5-benzisoxalylmethoxycarbonyl, 4-acetoxybenzyloxycarbonyl,2,2,2-trichloroethoxycarbonyl, 2-ethynyl-2-propoxycarbonyl,cyclopropylmethoxycarbonyl, 4-(decyloxyl)benzyloxycarbonyl,isobornyloxycarbonyl, 1-piperidyloxycarbonyl and 9-fluorenylmethylcarbonate.

In some embodiments, the protecting agent for amine protection isselected from the group consisting of benzylchloroformate,p-nitro-chlorobenzylformate, ethylchloroformate,di-tert-butyl-dicarbonate, triphenylmethyl chloride andmethoxytriphenylmethyl chloride. In a preferred embodiment, theprotecting group is benzyloxycarbonyl, installed by the protecting agentbenzyloxychloroformate.

Thiol protecting groups are well known to those skilled in the art. See,for example, Greene and Wuts (1999), Chapter 6. These protecting groupscan be installed via protecting agents well known to those of skill inthe art. Removal of these groups is also well known to those of skill inthe art.

In some embodiments, a thiol protecting group may be selected from thegroup consisting of acetamidomethyl, benzamidomethyl, 1-ethoxyethyl,benzoyl, triphenylmethyl, t-butyl, benzyl, adamantyl, cyanoethyl, acetyland trifluoroacetyl.

In some embodiments, the protecting agent for thiol protection isselected from the group consisting of an alkyl halide, a benzyl halide,a benzoyl halide, a sulfonyl halide, a triphenylmethyl halide, amethoxytriphenylmethyl halide and cysteine. Non-limiting examples ofthese protecting agents include ethyl halides, propyl halides and acetylhalides. Halides may comprise chloro, bromo or iodo, for example. In apreferred embodiment, the protecting group is benzyl, installed by theprotecting agent benzyl chloride.

Hydroxy (or alcohol) protecting groups are well known to those skilledin the art. See, for example, Greene and Wuts (1999), Chapter 2. Theseprotecting groups can be installed via protecting agents well known tothose of skill in the art. Removal of these groups is also well known tothose of skill in the art.

A suitable hydroxy protecting group may be selected from the groupconsisting of esters or ethers. Esters such as acetate, benzoyl,tert-butylcarbonyl and trifluoroacetyl groups are removable by acidic orbasic conditions. Ethers such as methoxy, ethoxy and tri-benzylmethylare removable by stronger acidic or basic conditions. A preferredprotecting group is an acetate ester.

Carbonyl protecting groups are well known to those skilled in the art.See, for example, Greene and Wuts (1999), Chapter 4. Such protectinggroups may protect, for example, ketones or aldehydes, or the carbonylpresent in esters, amides, esters and the like. These protecting groupscan be installed via protecting agents well known to those of skill inthe art. Removal of these groups is also well known to those of skill inthe art.

In some embodiments, a carbonyl protecting group may be selected fromthe group consisting of dimethylacetal, dimethylketal,diisopropylacetal, diisopropylketal, enamines and enol ethers.

Carboxylic acid protecting groups are well known to those skilled in theart. See, for example, Greene and Wuts (1999), Chapter 5. Removal ofthese groups is also well known to those of skill in the art.

A suitable carboxylic acid protecting group may be selected from thegroup consisting of amides or esters, for example. Amides such assulfonamide, para-nitroaniline, benzylamide and benzolyamide may behydrolyzed in acidic conditions. Esters such as methyl ester, ethylester and benzyl ester may be hydrolyzed by acidic or basic conditions.A preferred protecting group is an amide.

C. Metal Ions

As set forth above, certain embodiments of the present invention pertainto compositions that will function to chelate one or more metal ions.The targeting ligands of the present invention may also participate inchelating one or more metal ions. A “metal ion” is defined herein torefer to a metal ion that is capable of forming a bond, such as anon-covalent bond, with one or more atoms or molecules. The otheratom(s) or molecule(s) may be negatively charged.

Any metal ion known to those of ordinary skill in the art iscontemplated for inclusion in the compositions of the present invention.One of ordinary skill in the art would be familiar with the metal ionsand their application(s). In some embodiments, the metal ion may beselected from the group consisting of Tc-99m, Cu-60, Cu-61, Cu-62,Cu-67, In-111, Tl-201, Ga-67, Ga-68, As-72, Re-186, Re-187, Re-188,Ho-166, Y-90, Sm-153, Sr-89, Gd-157, Bi-212, Bi-213, Fe-56, Mn-55,Lu-177, a iron ion, a arsenic ion, a selenium ion, a thallium ion, amanganese ion, a cobalt ion, a platinum ion, a rhenium ion, a calciumion and a rhodium ion. For example, the metal ion may be a radionuclide.A radionuclide is an isotope of artificial or natural origin thatexhibits radioactivity. In some embodiments, the radionuclide isselected from the group consisting of ^(99m)Tc, ¹⁸⁸Re, ¹⁸⁶Re, ¹⁵³Sm,¹⁶⁶Ho, ⁹⁰Y, ⁸⁹Sr, ⁶⁷Ga, ⁶⁸Ga, ¹¹¹In, ¹⁷⁸Gd, ⁵⁵Fe, ²²⁵Ac, ²¹²Bi, ²¹¹At,⁴⁵Ti, ⁶⁰Cu, ⁶¹Cu, ⁶⁷Cu, and ⁶⁴Cu. In preferred embodiments, the metalion is rhenium or a radionuclide such as ^(99m)Tc, ¹⁸⁸Re, or ⁶⁸Ga. Asdescribed below, a reducing agent may need to accompany one of theradionuclides, such as ^(99m)Tc. Non-limiting examples of such reducingagents include a dithionite ion, a stannous ion and a ferrous ion.

Due to better imaging characteristics and lower price, attempts havebeen made to replace the ¹²³I, ¹³¹I, ⁶⁷Ga and ¹¹¹In labeled compoundswith corresponding ^(99m)Tc labeled compounds when possible. Due tofavorable physical characteristics as well as extremely low price($0.21/mCi), ^(99m)Tc has been preferred to label radiopharmaceuticals.

A number of factors must be considered for optimal radioimaging inhumans. To maximize the efficiency of detection, a metal ion that emitsgamma energy in the 100 to 200 keV range is preferred. A “gamma emitter”is herein defined as an agent that emits gamma energy of any range. Oneof ordinary skill in the art would be familiar with the various metalions that are gamma emitters. To minimize the absorbed radiation dose tothe patient, the physical half-life of the radionuclide should be asshort as the imaging procedure will allow. To allow for examinations tobe performed on any day and at any time of the day, it is advantageousto have a source of the radionuclide always available at the clinicalsite. ^(99m)Tc is a preferred radionuclide because it emits gammaradiation at 140 keV, it has a physical half-life of 6 hours, and it isreadily available on-site using a molybdenum-99/technetium-99mgenerator. One of ordinary skill in the art would be familiar withmethods to determine optimal radioimaging in humans.

In certain particular embodiments of the present invention, the metalion is a therapeutic metal ion. For example, in some embodiments, themetal ion is a therapeutic radionuclide that is a beta-emitter. Asherein defined, a beta emitter is any agent that emits beta energy ofany range. Examples of beta-emitters include Re-188, Re-187, Re-186,Ho-166, Y-90, Bi-212, Bi-213, and Sn-153. The beta-emitters may or maynot also be gamma-emitters. One of ordinary skill in the art would befamiliar with the use of beta-emitters in the treatment ofhyperproliferative disease, such as cancer.

In further embodiments of the compositions of the present invention, themetal ion is a therapeutic metal ion that is not a beta emitter or agamma emitter. For example, the therapeutic metal ion may be platinum,cobalt, copper, arsenic, selenium, calcium or thallium. Compositionsincluding these therapeutic metal ions may be applied in methodsdirected to the treatment of diseases such as hyperproliferativediseases, cardiovascular disease, infections, and inflammation. Examplesof hyperproliferative diseases include cancers. Methods of performingdual chemotherapy and radiation therapy that involve the compositions ofthe present invention are discussed in greater detail below.

D. Targeting Ligands

A “targeting ligand” is defined herein to be a molecule or part of amolecule that binds with specificity to another molecule. One ofordinary skill in the art would be familiar with the numerous agentsthat can be employed as targeting ligands in the context of the presentinvention.

Examples of targeting ligands include disease cell cycle targetingcompounds, angiogenesis targeting ligands, tumor apoptosis targetingligands, disease receptor targeting ligands, gene expression markers,drug-based ligands, antimicrobials, tumor hypoxia targeting ligands, anantisense molecule, an agent that mimics glucose, amifostine,angiostatin, EGF receptor ligands, capecitabine, COX-2 inhibitors,deoxycytidine, fullerene, herceptin, human serum albumin, lactose,leuteinizing hormone, pyridoxal, quinazoline, thalidomide, transferrin,and trimethyl lysine.

In further embodiments of the present invention, the targeting ligand isan antibody. Any antibody is contemplated as a targeting ligand in thecontext of the present invention. For example, the antibody may be amonoclonal antibody. One of ordinary skill in the art would be familiarwith monoclonal antibodies, methods of preparation of monoclonalantibodies, and methods of use of monoclonal antibodies as ligands. Incertain embodiments of the present invention, the monoclonal antibody isan antibody directed against a tumor marker. In some embodiments, themonoclonal antibody is monoclonal antibody C225, monoclonal antibodyCD31, or monoclonal antibody CD40.

A single targeting ligand, or more than one such targeting ligand, maybe conjugated to a chelator of the present invention. In theseembodiments, any number of targeting ligands may be conjugated to thechelators set forth herein. In certain embodiments, a conjugate of thepresent invention may comprise a single targeting ligand. In otherembodiments, a conjugate may comprise only two targeting ligands. Infurther embodiments, a targeting ligand may comprise three or moretargeting ligands. In any situation where a conjugate comprises two ormore targeting ligands, the targeting ligands may be the same ordifferent.

The targeting ligands can be bound to the chelator in any manner,including for example covalent bonds, ionic bonds and hydrogen bonds.For example, the targeting ligand may be bound to the chelator in anamide linkage, an ester linkage, or a carbon-carbon bond linkage of anylength. If two or more targeting ligands are bound to a chelator, themodes of binding may be the same or different. In other embodiments, thelinkage comprises a linker. Non-limiting examples of such linkersinclude peptides, glutamic acid, aspartic acid, bromo ethylacetate,ethylene diamine, lysine and any combination of one or more of thesegroups. One of ordinary skill in the art would be familiar with thechemistry of these agents, and methods to conjugate these agents asligands to the chelators of the claimed invention. Methods of synthesisof the compounds of the present invention, including modes ofconjugation, are discussed in detail below.

Information pertaining to targeting ligands and conjugation withcompounds is provided in U.S. Pat. No. 6,692,724, U.S. patentapplication Ser. No. 09/599,152, U.S. patent application Ser. No.10/627,763, U.S. patent application Ser. No. 10/672,142, U.S. patentapplication Ser. No. 10/703,405, and U.S. patent application Ser. No.10/732,919, each of which is herein specifically incorporated byreference in their entirety for this section of the specification andall other sections of the specification.

In some embodiments of the compositions of the present invention, thetargeting ligand is a tissue-specific ligand, which is conjugated to thechelator. A “tissue-specific ligand” is defined herein to refer to amolecule or a part of a molecule that can bind or attach to one or moretissues. The binding may be by any mechanism of binding known to thoseof ordinary skill in the art. Examples include therapeutic agents,antimetabolites, apoptotic agents, bioreductive agents, signaltransductive therapeutic agents, receptor responsive agents, or cellcycle specific agents. The tissue may be any type of tissue, such as acell. For example, the cell may be the cell of a subject, such as acancer cell. In certain embodiments, the tissue-targeting ligand is atissue-targeting amino acid sequence that is conjugated to a chelatorthat is capable of binding to a metal ion.

Representative examples of targeting ligands are discussed below.

1. Drugs

In some embodiments of the compositions of the present invention, atargeting ligand is a drug, or “therapeutic ligand,” which is definedherein to refer to any therapeutic agent. A “therapeutic agent” or“drug” is defined herein to include any compound or substance that canbe administered to a subject, or contacted with a cell or tissue, forthe purpose of treating a disease or disorder, or preventing a diseaseor disorder, or treating or preventing an alteration or disruption of anormal physiologic process. For example, the therapeutic ligand may bean anti-cancer moiety, such as a chemotherapeutic agent. In certainembodiments of the present invention, the therapeutic ligand is atherapeutic amino acid sequence that is conjugated to the therapeuticamino acid sequence. Such conjugates are discussed further in otherparts of this specification.

a. Chemotherapeutic Agents

Examples of anti-cancer ligands include any chemotherapeutic agent knownto those of ordinary skill in the art. Examples of such chemotherapeuticagents include, but are not limited to, cisplatin (CDDP), carboplatin,procarbazine, mechlorethamine, cyclophosphamide, camptothecin,ifosfamide, melphalan, chlorambucil, busulfan, nitrosurea, dactinomycin,daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide(VP16), tamoxifen, raloxifene, estrogen receptor binding agents, taxol,gemcitabien, navelbine, farnesyl-protein transferase inhibitors,transplatinum, 5-fluorouracil, vincristin, vinblastin and methotrexate,or any analog or derivative variant of the foregoing. In certainparticular embodiments, the anti-cancer ligand is methotrexate.

A wide variety of chemotherapeutic agents may be used in accordance withthe present invention. The term “chemotherapy” refers to the use ofdrugs to treat cancer. A “chemotherapeutic agent” is used to connote acompound or composition that is administered in the treatment of cancer.These agents or drugs are categorized by their mode of activity within acell, for example, whether and at what stage they affect the cell cycle.Alternatively, an agent may be characterized based on its ability todirectly cross-link DNA, to intercalate into DNA, or to inducechromosomal and mitotic aberrations by affecting nucleic acid synthesis.Most chemotherapeutic agents fall into the following categories:alkylating agents, antimetabolites, antitumor antibiotics, mitoticinhibitors, and nitrosoureas.

Examples of chemotherapeutic agents include alkylating agents such asthiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan,improsulfan and piposulfan; aziridines such as benzodepa, carboquone,meturedepa, and uredepa; ethylenimines and methylamelamines includingaltretamine, triethylenemelamine, trietylenephosphoramide,triethiylenethiophosphoramide and trimethylolomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analogue topotecan); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); cryptophycins (particularly cryptophycin 1 and cryptophycin8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin;spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimustine; antibiotics such as the enediyne antibiotics (e.g.,calicheamicin, especially calicheamicin gammalI and calicheamicinomegaI1; dynemicin, including dynemicin A); bisphosphonates, such asclodronate; an esperamicin; as well as neocarzinostatin chromophore andrelated chromoprotein enediyne antibiotic chromophores, aclacinomysins,actinomycin, authramycin, azaserine, bleomycins, cactinomycin,carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin(including morpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolicacid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin,quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexateand 5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharidecomplex); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonicacid; triaziquone; 2,2′,2″-trichlorotriethylamine; trichothecenes(especially T-2 toxin, verracurin A, roridin A and anguidine); urethan;vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol;pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide;thiotepa; taxoids, e.g., paclitaxel and doxetaxel; chlorambucil;gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinumcoordination complexes such as cisplatin, oxaliplatin and carboplatin;vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone;vincristine; vinorelbine; novantrone; teniposide; edatrexate;daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11);topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO);retinoids such as retinoic acid; capecitabine; and pharmaceuticallyacceptable salts, acids or derivatives of any of the above.

Also included in this definition are anti-hormonal agents that act toregulate or inhibit hormone action on tumors such as anti-estrogens andselective estrogen receptor modulators (SERMs), including, for example,tamoxifen, raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene,keoxifene, LY117018, onapristone, and toremifene; aromatase inhibitorsthat inhibit the enzyme aromatase, which regulates estrogen productionin the adrenal glands, such as, for example, 4(5)-imidazoles,aminoglutethimide, megestrol acetate, exemestane, formestanie,fadrozole, vorozole, letrozole, and anastrozole; and anti-androgens suchas flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; aswell as troxacitabine (a 1,3-dioxolane nucleoside cytosine analog);antisense oligonucleotides, particularly those which inhibit expressionof genes in signaling pathways implicated in abherant cellproliferation, such as, for example, PKC-alpha, Ralf and H-Ras;ribozymes such as a VEGF expression inhibitor and a HER2 expressioninhibitor; vaccines such as gene therapy vaccines and pharmaceuticallyacceptable salts, acids or derivatives of any of the above.

Additional examples of anti-cancer agents include those drugs of choicefor cancer chemotherapy listed in Table 1:

TABLE 1 Drugs of Choice for Cancer Chemotherapy The tables that followlist drugs used for treatment of cancer in the USA and Canada and theirmajor adverse effects. The Drugs of Choice listing based on the opinionsof Medical Letter consultants. Some drugs are listed for indications forwhich they have not been approved by the U.S. Food and DrugAdministration. Anti-cancer drugs and their adverse effects follow. Forpurposes of the present invention, these lists are meant to be exemplaryand not exhaustive. DRUGS OF CHOICE Cancer Drugs of Choice Somealternatives Adrenocortical** Mitotane Doxorubicin, streptozocin,Cisplatin etoposide Bladder* Local: Instillation of BCG Instillation ofmitomycin, Systemic: Methotrexate + vinblastine + doxorubicin +cisplatin doxorubicin or thiotape (MVAC) Pacitaxel, substitution ofCisplatin + Methotrexate + vinblastine carboplatin for cisplatin in(CMV) combinations Brain Anaplastic astrocytoma* Procarbazine +lomustine + vincristine Carmustine, Cisplatin Anaplastic oligodendro-Procarbazine + lomustine + vincristine Carmustine, Cisplatin Glioma*Glioblastoma** Carmustine or lomustine Procarbazine, cisplatinMedulloblastoma Vincristine + carmustine ± mechlorethamine ±methotrexate Etoposide Mechiorethamine + vincristine + procarbazine +prednisone (MOPP) Vincristine + cisplatin ± cyclophosphamide Primarycentral nervous Methotrexate (high dose Intravenous and/or systemlymphoma Intrathecal) ± cytarabine (Intravenous and/or Intrathecal)Cyclophosphamide + Doxorubicin + vincristine + prednisone (CHOP) BreastAdjuvant¹: Cyclophosphamide + methotrexate + fluorouracil Paclitaxel;thiotepa + Doxorubicin + vinblastine; (CMF); mitomycin + vinblastine;Cyclophosphamide + Doxorubicin ± fluorouracil mitomycin + methotrexate +mitoxantrone; (AC or CAF); Tamoxifen fluorouracil Metastatic:Cyclophosphamide + methotrexate + fluorouracil by continuous infusion;(CMF) or Bone marrow transplant³ Cyclophosphamide + doxorubicin ±fluorouracil (AC or CAF) for receptor- negative and/orhormone-refractory; Tamoxifen for receptor-positive and/orhormone-sensitive² Cervix** Cisplatin Chlorambucil, vincristine,Ifosfamide with means fluorouracil, Doxorubicin, Bleomycin + ifosfamidewith means + cisplatin methotrexate, altretamine ChoriocarcinomaMethotrexate ± leucovorin Methotrexate + Dactinomycin dactinomycin +cyclophosphamide (MAC) Etoposide + methotrexate + dactinomycin +cyclophosphamide + vincristine Colorectal* Adjuvant colon⁴:Fluorouracil + levamisole; Hepatic metastases: fluorouracil + leucovorinIntrahepatic-arterial Metastatic: fluorouracil + leucovorin floxuridineMitomycin Embryonal rhabdomyosarcoma⁵ Vincristine + dactinomycin ±cyclophosphamide Same + Doxorubicin Vincristine + ifosfamide withmeans + etoposide Endometrial** Megastrol or another progestinfluorouracil, tamoxifen, Doxorubicin + cisplatin ± cyclophosphamidealtretamine Esophageal* Cisplatin + fluorouracil Doxorubicin,methotraxate, mitomycin Ewing's sarcoma⁵ Cyclophosphamide (or ifosfamidewith CAV + etoposide means) + Doxorubicin + vincristine (CAV) ±dactinomycin Gastric** Fluorouracil ± leucovorin Cisplatin, Doxorubicin,etoposide, methotrexate + leucovorin, mitomycin Head and neck squamouscell* Cisplatin + fluorouracil Bleomycin, carboplatin, Methotrexatepaclitaxel Islet cell** Streptozocin + Doxorubicin Streptozocin +fluorouracil; chlorozotocin^(†); octreotide Kaposi's sarcoma*(Aids-related) Etoposide or interferon alfa or vinblastine Vincristine,Doxorubicin, Doxorubicin + bleomycin + vincristine or bleomycinvinblastine (ABV) Leukemia Acute lymphocytic leukemia Induction:Vincristine + Induction: same ± high- (ALL)⁶ prednisone + asparaginase ±daunorubicin dose methotrexate ± cytarabine; CNS prophylaxis:Intrathecal methotrexate ± systemic high-dose methotrexate withpegaspargase leucovorin ± Intrathecal cytarabine ± Intrathecal insteadof asparaginese hydrocortisone Teniposide or etoposide Maintenance:Methotrexate + mercaptopurine High-dose cytarabine Bone marrowtransplant.³ ⁷ Maintenance: same + periodic vincristine + prednisoneAcute myeloid leukemia (AML)⁸ Induction: Cytarahine + eitherdaunorubicin Cytarabine + mitoxentrone or idarubicin High-dosecytarabine Post Induction: High-dose cytarabine ± other drugs such asetoposide Bone marrow transplant³. Chronic lymphocytic leukemiaChlorambucil ± prednisone Cladribine, (CLL) Fludarabin cyclophosphamide,pentostatin, vincristine, Doxorubicin Chronic myeloid leukemia (CML)⁹Chronic phase Bone marrow transplant³ Busulfan Interferon alfaHydroxyurea Accelerated¹⁰ Bone marrow transplant³ Hydroxyures, busulfanBlast crisis¹¹ Lymphoid: Vincristine + prednisone + L- Tretinoln^(†)asparaginase + intrathecal methotrexate (± maintenance Amsecrine,^(†)azacitidine with methotrexate + 8- Vincristine ± plicamycinmercaptopurine) Hairy cell Leukemia Pentostatin or cladribine Interferonalfa, chlorambucil, fludarabin Liver** Doxorubicin Intrahepatic-arterialFluorouracil floxuridine or claplatin Lung, small cell (cat cell)Cisplatin + etoposide (PE) Ifosfamide with means + carboplatin +etoposide Cyclophosphamide + doxorubicin + vincristine (ICE) (CAV) Dailyoral etoposide PE alternated with CAV Etoposide + ifosfamideCyclophosphamide + etoposide + cisplatin with means + claplatin (CEP)(VIP Doxorubicin + cyclophosphamide + etoposide Paclitaxel (ACE) LungCisplatin + etoposide Cisplatin + fluorouracil + leucovorin (non-smallcell)** Cisplatin + Vinblastine ± mitomycin Carboplatin + paclitaxelCisplatin + vincristine Lymphomas Hodgkin's¹¹ Doxorubicin + bleomycin +vinblastine + dacarbazine Mechlorethamine + vincristine + (ABVD)procarbazine + prednisone (MOPP) ABVD alternated with MOPPChlorambusil + vinblastine + Mechlorethamine + vincristine +procarbazine + prednisone ± carmustine procarbazine (± prednisone) +Etoposide + doxorubicin + bleomycin + vinblastine vinblastine +doxorubicin (MOP[P]-ABV) Bone marrow transplant³ Non-Hodgkin'sCyclophosphamide + vincristine + methotrexate Ifosfamide with meansBurkitt's lymphoma Cyclophosphamide + high-dose cytarabine ±methotrexate Cyclophosphamide + doxorubicin + with leutovorinvincristine + prednisone (CHOP) Intrathecal methotrexate or cytarabineDiffuse large-cell lymphoma Cyclophosphamide + doxorubicin +vincristine + prednisone Dexamethasone (CHOP) sometimes substituted forprednisone Other combination regimens, which may include methotrexate,etoposide, cytarabine, bleomycin, procarbazine, ifosfamide andmitoxantrone Bone marrow transplant³ Follicular lymphomaCyclophosphamide or chlorambusil Same ± vincristine and prednisone, ±etoposide Interferon alfa, cladribine, fludarabin Bone marrowtransplant³ Cyclophosphamide + doxorubicin + vincristine + prednisone(CHOP) Melanoma** Interferon Alfa Carmustine, lomustine, Dacarbazinecisplatin Dacarbazine + clapletin + carmustine + tamoxifen AldesleukinMycosis fungoides* PUVA (psoralen + ultraviolet A) Isotretinoin, topicalMechlorethamine (topical) carmustine, pentosistin, Interferon alfafludarabin, cladribine, Electron beam radiotherapy photopheresis (extra-Methotrexate corporeal photochemitherapy), chemotherapy as in non-Hodgkin's lymphoma Myloma* Melphalan (or cyclophosphamide) + prednisoneInterferon alfa Melphalan + carmustine + Bone marrow transplant³cyclophosphamide + prednisone + vincristine High-dose dexamethasoneDexamethasone + doxorubicin + vincristine (VAD) Vincristine +carmustine + doxorubicin + prednisone (VBAP) Neuroblastoma*Doxorubicin + cyclophosphamide + cisplatin + teniposide Carboplatin,etoposide or etoposide Bone marrow transplant³ doxorubicin +cyclophosphamide Claplatin + cyclophosphamide Osteogenic sarcoma⁵Doxorubicin + cisplatin ± etoposide ± ifosfamide Ifosfamide with means,etoposide, carboplatin, high-dose methotrexate with leucovorinCyclophosphamide + etoposide Ovary Cisplatin (or carboplatin) +paclitaxel Ifosfamide with means, Cisplatin (or carboplatin) +cyclophosphamide paclitaxel, tamoxifen, (CP) ± doxorubicin melphalan,altretamine (CAP) Pancreatic** Fluorouracil ± leucovorin ProstateLeuprolide + flutamide Estramustine + vinblastine, aminoglutethimide +hydrocortisone, estramustine + etoposide, diethylstilbestrol, nilutamideRenal** Aldesleukin Vinblastine, floxuridine Inteferon alfaRetinoblastoma^(5*) Doxorubicin + cyclophosphamide + Carboplatin,etoposide, cisplatin + etoposide + vincristine Ifosfamide with meansSarcomas, soft tissue, adult* Doxorubicin + dacarbazine +cyclophosphamide + Ifosfamide Mitornyeln + doxorubicin + cisplatin withmeans Vincristine, etoposide Testicular Cisplatin + etoposide +bleomycin (PEB) Vinblastine (or etoposide) + Ifosfamide with means +cisplatin (VIP) Bone marrow transplant³ Wilms' tumor⁵ Dactinomycin +vincristine + doxorubicin + cyclophosphamide Ifosfamide with means,etoposide, carboplatin *Chemotherapy has only moderate activity.**Chemotherapy has only minor activity. ¹Tamoxifen with or withoutchemotherapy is generally recommended for postmenopausalestrogen-receptor-positive, mode-positive patients and chemotherapy withor without tamoxifen for premenopausal mode-positive patients. Adjuvanttreatment with chemotherapy and/or tamoxifen is recommended formode-negative patients with larger tumors or other adverse prognosticindicators. ²Megastrol and other hormonal agents may be effective insome patients with tamoxifen fails. ³After high-dose chemotherapy(Medical Letter, 34: 79, 1982). ⁴For rectal cancer, postoperativeadjuvant treatment with fluorouracil plus radiation, preceded andfollowed by treatment with fluorouracil alone. ⁵Drugs have majoractivity only when combined with surgical resection, radiotherapy orboth. ^(†)Available in the USA only for investigational use. ⁶High-riskpatients (e.g., high counts, cytogenetic abnormalities, adults) mayrequire additional drugs for induction, maintenance and“intensificiation” (use of additional drugs after achievement ofremission). Additional drugs include cyclophosphamida, mitoxantrone andthloguanine. The results of one large controlled trial in the UnitedKingdom suggest that intensificiation may improve survival in allchildren with ALL (Chasselle et al, 1995). ⁷Patients with a poorprognosis initially or those who relapse after remission. ⁸Some patientswith acute promyelocytic leukemia have had complete responses totratinoin. Such treatment can cause a toxic syndrome characterizedprimarily by fever and respiratory distress (Warrell, Jr et al, 1993).⁹Allogeneic HLA-identical sibling bone marrow transplantation can cure40% to 70% of patients with CML in chronic phase, 18% to 28% of patientswith accelerated phase CML, and <15% patients in blast crisis.Disease-free survival after bone marrow transplantations adverselyinfluenced by age >50 years, duration of disease >3 years fromdiagnosis, and use of one-antigen-mismatched or matched-unrelated donormarrow. Interferon also may be curative in patients with chronic phaseCML who achieve a complete cytogenetic response (about 10%); it is thetreatment of choice for patents >80 years old with newly diagnosedchronic phase CML and for all patients who are not candidates for anallgensic bone marrow transplant. Chemotherapy alone is palliative. ¹⁰Ifa second chronic phase is achieved with any of these combinations,allogeneic bone marrow transplant should be considered. Bone marrowtransplant in second chronic phase may be curative for 30% to 35% ofpatients with CML. ¹¹Limited-stage Hodgkin's disease (stages 1 and 2) iscurable by radiotherapy. Disseminated disease (stages 3b and 4) requirechemotherapy. Some intermediate stages and selected clinical situationsmay benefit from both. + Available in the USA only for investigationaluse.

b. Cardiovascular Drugs

A “cardiovascular drug” is defined herein to refer to any therapeuticagent that can be applied in the treatment or prevention of a disease ofthe heart and/or blood vessels.

In certain embodiments, the cardiovascular drug is an agent that lowersthe concentration of one of more blood lipids and/or lipoproteins, knownherein as an “antihyperlipoproteinemic,” which can be applied in thetreatment of atherosclerosis and thickenings or blockages of vasculartissues. Examples include an aryloxyalkanoic/fibric acid derivative, aresin/bile acid sequesterant, a HMG CoA reductase inhibitor, a nicotinicacid derivative, a thyroid hormone or thyroid hormone analog, amiscellaneous agent or a combination thereof. Non-limiting examples ofaryloxyalkanoic/fibric acid derivatives include beclobrate,benzafibrate, binifibrate, ciprofibrate, clinofibrate, clofibrate(atromide-S), clofibric acid, etofibrate, fenofibrate, gemfibrozil(lobid), nicofibrate, pirifibrate, ronifibrate, simfibrate andtheofibrate. Non-limiting examples of resins/bile acid sequestrantsinclude cholestyramine (cholybar, questran), colestipol (colestid) andpolidexide. Non-limiting examples of HMG CoA reductase inhibitorsinclude lovastatin (mevacor), pravastatin (pravochol) or simvastatin(zocor). Non-limiting examples of nicotinic acid derivatives includenicotinate, acepimox, niceritrol, nicoclonate, nicomol and oxiniacicacid. Non-limiting examples of thyroid hormones and analogs thereofinclude etoroxate, thyropropic acid and thyroxine. Non-limiting examplesof miscellaneous antihyperlipoproteinemics include acifran, azacosterol,benfluorex, β-benzalbutyramide, camitine, chondroitin sulfate,clomestrone, detaxtran, dextran sulfate sodium,5,8,11,14,17-eicosapentaenoic acid, eritadenine, furazabol, meglutol,melinamide, mytatrienediol, ornithine, γ-oryzanol, pantethine,pentaerythritol tetraacetate, α-phenylbutyramide, pirozadil, probucol(lorelco), β-sitosterol, sultosilic acid-piperazine salt, tiadenol,triparanol and xenbucin.

Non-limiting examples of an antiarteriosclerotic include pyridinolcarbamate.

In certain embodiments, the cardiovascular drug is an agent that aids inthe removal or prevention of blood clots. Non-limiting examples ofantithrombotic and/or fibrinolytic agents include anticoagulants,anticoagulant antagonists, antiplatelet agents, thrombolytic agents,thrombolytic agent antagonists or combinations thereof. Examples ofantithrombotic agents include aspirin and wafarin (coumadin. Examples ofanticoagulant include acenocoumarol, ancrod, anisindione, bromindione,clorindione, coumetarol, cyclocumarol, dextran sulfate sodium,dicumarol, diphenadione, ethyl biscoumacetate, ethylidene dicoumarol,fluindione, heparin, hirudin, lyapolate sodium, oxazidione, pentosanpolysulfate, phenindione, phenprocoumon, phosvitin, picotamide,tioclomarol and warfarin. Non-limiting examples of antiplatelet agentsinclude aspirin, a dextran, dipyridamole (persantin), heparin,sulfinpyranone (anturane) and ticlopidine (ticlid). Non-limitingexamples of thrombolytic agents include tissue plasminogen activator(activase), plasmin, pro-urokinase, urokinase (abbokinase) streptokinase(streptase), antistreplase/APSAC (eminase).

In some embodiments, the cardiovascular drug is a blood coagulant.Non-limiting examples of a blood coagulation promoting agent includethrombolytic agent antagonists and anticoagulant antagonists.Non-limiting examples of anticoagulant antagonists include protamine andvitamin K1.

Non-limiting examples of thrombolytic agent antagonists includeaminocaproic acid (amicar) and tranexamic acid (amstat). Non-limitingexamples of antithrombotics include anagrelide, argatroban, cilostazol,daltroban, defibrotide, enoxaparin, fraxiparine, indobufen, lamoparan,ozagrel, picotamide, plafibride, tedelparin, ticlopidine and triflusal.

The cardiovascular drug may be an antiarrythmic agent. Non-limitingexamples of antiarrhythmic agents include Class I antiarrythmic agents(sodium channel blockers), Class II antiarrythmic agents(beta-adrenergic blockers), Class II antiarrythmic agents(repolarization prolonging drugs), Class IV antiarrhythmic agents(calcium channel blockers) and miscellaneous antiarrythmic agents.Non-limiting examples of sodium channel blockers include Class IA, ClassIB and Class IC antiarrhythmic agents. Non-limiting examples of Class IAantiarrhythmic agents include dispyramide (norpace), procainamide(pronestyl) and quinidine (quinidex). Non-limiting examples of Class IBantiarrhythmic agents include lidocaine (xylocaine), tocamide (tonocard)and mexiletine (mexitil). Non-limiting examples of Class ICantiarrhythmic agents include encamide (enkaid) and flecaimide(tambocor). Non-limiting examples of a beta blocker, otherwise known asa β-adrenergic blocker, a β-adrenergic antagonist or a Class IIantiarrhythmic agent, include acebutolol (sectral), alprenolol,amosulalol, arotinolol, atenolol, befunolol, betaxolol, bevantolol,bisoprolol, bopindolol, bucumolol, bufetolol, bufuralol, bunitrolol,bupranolol, butidrine hydrochloride, butofilolol, carazolol, carteolol,carvedilol, celiprolol, cetamolol, cloranolol, dilevalol, epanolol,esmolol (brevibloc), indenolol, labetalol, levobunolol, mepindolol,metipranolol, metoprolol, moprolol, nadolol, nadoxolol, nifenalol,nipradilol, oxprenolol, penbutolol, pindolol, practolol, pronethalol,propranolol (inderal), sotalol (betapace), sulfinalol, talinolol,tertatolol, timolol, toliprolol and xibinolol. In certain aspects, thebeta blocker comprises an aryloxypropanolamine derivative. Non-limitingexamples of aryloxypropanolamine derivatives include acebutolol,alprenolol, arotinolol, atenolol, betaxolol, bevantolol, bisoprolol,bopindolol, bunitrolol, butofilolol, carazolol, carteolol, carvedilol,celiprolol, cetamolol, epanolol, indenolol, mepindolol, metipranolol,metoprolol, moprolol, nadolol, nipradilol, oxprenolol, penbutolol,pindolol, propranolol, talinolol, tertatolol, timolol and toliprolol.Non-limiting examples of an agent that prolong repolarization, alsoknown as a Class III antiarrhythmic agent, include amiodarone(cordarone) and sotalol (betapace). Non-limiting examples of a calciumchannel blocker, otherwise known as a Class IV antiarrythmic agent,include an arylalkylamine (e.g., bepridile, diltiazem, fendiline,gallopamil, phenylamine, terodiline, verapamil), a dihydropyridinederivative (felodipine, isradipine, nicardipine, nifedipine, nimodipine,nisoldipine, nitrendipine) a piperazine derivative (e.g., cinnarizine,flunarizine, lidoflazine) or a miscellaneous calcium channel blockersuch as bencyclane, etafenone, magnesium, mibefradil or perhexyline. Incertain embodiments a calcium channel blocker comprises a long-actingdihydropyridine (nifedipine-type) calcium antagonist. Non-limitingexamples of miscellaneous antiarrhymic agents include adenosine(adenocard), digoxin (lanoxin), acetamide, ajmaline, amoproxan,aprindine, bretylium tosylate, bunaftine, butobendine, capobenic acid,cifenline, diisopyramide, hydroquinidine, indecamide, ipratropiumbromide, lidocaine, lorajmine, lorcamide, meobentine, moricizine,pirmenol, prajmaline, propafenone, pyrinoline, quinidinepolygalacturonate, quinidine sulfate and viquidil.

Other examples of cardiovascular drugs include antihypertensive agents.Non-limiting examples of antihypertensive agents include sympatholytic,alpha/beta blockers, alpha blockers, anti-angiotensin II agents, betablockers, calcium channel blockers, vasodilators and miscellaneousantihypertensives. Non-limiting examples of an alpha blocker, also knownas an α-adrenergic blocker or an α-adrenergic antagonist, includeamosulalol, arotinolol, dapiprazole, doxazocin, ergoloid mesylates,fenspiride, indoramin, labetalol, nicergoline, prazosin, terazosin,tolazoline, trimazosin and yohimbine. In certain embodiments, an alphablocker may comprise a quinazoline derivative. Non-limiting examples ofquinazoline derivatives include alfuzosin, bunazosin, doxazocin,prazosin, terazosin and trimazosin. In certain embodiments, anantihypertensive agent is both an alpha and beta adrenergic antagonist.Non-limiting examples of an alpha/beta blocker comprise labetalol(normodyne, trandate). Non-limiting examples of anti-angiotension IIagents include angiotensin converting enzyme inhibitors and angiotensionII receptor antagonists. Non-limiting examples of angiotensionconverting enzyme inhibitors (ACE inhibitors) include alacepril,enalapril (vasotec), captopril, cilazapril, delapril, enelaprilat,fosinopril, lisinopril, moveltopril, perindopril, quinapril andramipril. Non-limiting examples of an angiotensin II receptor blocker,also known as an angiotension II receptor antagonist, an ANG receptorblocker or an ANG-II type-1 receptor blocker (ARBS), includeangiocandesartan, eprosartan, irbesartan, losartan and valsartan.Non-limiting examples of a sympatholytic include a centrally actingsympatholytic or a peripherally acting sympatholytic. Non-limitingexamples of a centrally acting sympatholytic, also known as an centralnervous system (CNS) sympatholytic, include clonidine (catapres),guanabenz (wytensin) guanfacine (tenex) and methyldopa (aldomet).Non-limiting examples of a peripherally acting sympatholytic include aganglion blocking agent, an adrenergic neuron blocking agent, aβ-adrenergic blocking agent or a alpha1-adrenergic blocking agent.Non-limiting examples of a ganglion blocking agent include mecamylamine(inversine) and trimethaphan (arfonad). Non-limiting of an adrenergicneuron blocking agent include guanethidine (ismelin) and reserpine(serpasil). Non-limiting examples of a β-adrenergic blocker includeacenitolol (sectral), atenolol (tenormin), betaxolol (kerlone),carteolol (cartrol), labetalol (normodyne, trandate), metoprolol(lopressor), nadanol (corgard), penbutolol (levatol), pindolol (visken),propranolol (inderal) and timolol (blocadren). Non-limiting examples ofalpha1-adrenergic blocker include prazosin (minipress), doxazocin(cardura) and terazosin (hytrin). In certain embodiments acardiovasculator therapeutic agent may comprise a vasodilator (e.g., acerebral vasodilator, a coronary vasodilator or a peripheralvasodilator). In certain preferred embodiments, a vasodilator comprisesa coronary vasodilator. Non-limiting examples of a coronary vasodilatorinclude amotriphene, bendazol, benfurodil hemisuccinate, benziodarone,chloracizine, chromonar, clobenfurol, clonitrate, dilazep, dipyridamole,droprenilamine, efloxate, erythritol tetranitrane, etafenone, fendiline,floredil, ganglefene, herestrol bis(β-diethylaminoethyl ether),hexobendine, itramin tosylate, khellin, lidoflanine, mannitolhexanitrane, medibazine, nicorglycerin, pentaerythritol tetranitrate,pentrinitrol, perhexyline, pimefylline, trapidil, tricromyl,trimetazidine, trolnitrate phosphate and visnadine. In certain aspects,a vasodilator may comprise a chronic therapy vasodilator or ahypertensive emergency vasodilator. Non-limiting examples of a chronictherapy vasodilator include hydralazine (apresoline) and minoxidil(loniten). Non-limiting examples of a hypertensive emergency vasodilatorinclude nitroprusside (nipride), diazoxide (hyperstat IV), hydralazine(apresoline), minoxidil (loniten) and verapamil.

Non-limiting examples of miscellaneous antihypertensives includeajmaline, γ-aminobutyric acid, bufeniode, cicletainine, ciclosidomine, acryptenamine tannate, fenoldopam, flosequinan, ketanserin, mebutamate,mecamylamine, methyldopa, methyl 4-pyridyl ketone thiosemicarbazone,muzolimine, pargyline, pempidine, pinacidil, piperoxan, primaperone, aprotoveratrine, raubasine, rescimetol, rilmenidene, saralasin, sodiumnitroprusside, ticrynafen, trimethaphan camsylate, tyrosinase andurapidil.

In certain aspects, an antihypertensive may comprise an arylethanolaminederivative, a benzothiadiazine derivative, aN-carboxyalkyl(peptide/lactam) derivative, a dihydropyridine derivative,a guanidine derivative, a hydrazines/phthalazine, an imidazolederivative, a quanternary ammonium compound, a reserpine derivative or asulfonamide derivative. Non-limiting examples of arylethanolaminederivatives include amosulalol, bufuralol, dilevalol, labetalol,pronethalol, sotalol and sulfinalol. Non-limiting examples ofbenzothiadiazine derivatives include althizide, bendroflumethiazide,benzthiazide, benzylhydrochlorothiazide, buthiazide, chlorothiazide,chlorthalidone, cyclopenthiazide, cyclothiazide, diazoxide, epithiazide,ethiazide, fenquizone, hydrochlorothiazide, hydroflumethiazide,methyclothiazide, meticrane, metolazone, paraflutizide, polythiazide,tetrachlormethiazide and trichloromethiazide. Non-limiting examples ofN-carboxyalkyl(peptide/lactam) derivatives include alacepril, captopril,cilazapril, delapril, enalapril, enelaprilat, fosinopril, lisinopril,moveltipril, perindopril, Non-limiting examples of dihydropyridinederivatives include amlodipine, felodipine, isradipine, nicardipine,nifedipine, nilvadipine, nisoldipine and nitrendipine. Non-limitingexamples of guanidine derivatives include bethanidine, debrisoquin,guanabenz, guanacline, guanadrel, guanazodine, guanethidine, guanfacine,guanochlor, guanoxabenz and guanoxan. Non-limiting examples ofhydrazines/phthalazines include budralazine, cadralazine, dihydralazine,endralazine, hydracarbazine, hydralazine, pheniprazine, pildralazine andtodralazine. Non-limiting examples of imidazole derivatives includeclonidine, lofexidine, phentolamine, tiamenidine and tolonidine.Non-limiting examples of quanternary ammonium compounds includeazamethonium bromide, chlorisondamine chloride, hexamethonium,pentacynium bis(methylsulfate), pentamethonium bromide, pentoliniumtartrate, phenactropinium chloride and trimethidinium methosulfate.Non-limiting examples of reserpine derivatives include bietaserpine,deserpidine, rescinnamine, reserpine and syrosingopine. Non-limitingexamples of sulfonamide derivatives include ambuside, clopamide,furosemide, indapamide, quinethazone, tripamide and xipamide.

Other examples of cardiovascular drugs include vasopressors.Vasopressors generally are used to increase blood pressure during shock,which may occur during a surgical procedure. Non-limiting examples of avasopressor, also known as an antihypotensive, include amezinium methylsulfate, angiotensin amide, dimetofrine, dopamine, etifelmin, etilefrin,gepefrine, metaraminol, midodrine, norepinephrine, pholedrine andsynephrine.

Other examples of cardiovascular drugs include agents that can beapplied in the treatment or prevention of congestive heart failure.Non-limiting examples of agents for the treatment of congestive heartfailure include anti-angiotensin II agents, afterload-preload reductiontreatment, diuretics and inotropic agents. Examples of afterload-preloadreduction agents include hydralazine (apresoline) and isosorbidedinitrate (isordil, sorbitrate). Non-limiting examples of a diureticinclude a thiazide or benzothiadiazine derivative (e.g., althiazide,bendroflumethiazide, benzthiazide, benzylhydrochlorothiazide,buthiazide, chlorothiazide, chlorothiazide, chlorthalidone,cyclopenthiazide, epithiazide, ethiazide, ethiazide, fenquizone,hydrochlorothiazide, hydroflumethiazide, methyclothiazide, meticrane,metolazone, paraflutizide, polythiazide, tetrachloromethiazide,trichloromethiazide), an organomercurial (e.g., chlormerodrin,meralluride, mercamphamide, mercaptomerin sodium, mercumallylic acid,mercumatilin dodium, mercurous chloride, mersalyl), a pteridine (e.g.,furtherene, triamterene), purines (e.g., acefylline,7-morpholinomethyltheophylline, pamobrom, protheobromine, theobromine),steroids including aldosterone antagonists (e.g., canrenone, oleandrin,spironolactone), a sulfonamide derivative (e.g., acetazolamide,ambuside, azosemide, bumetamide, butazolamide, chloraminophenamide,clofenamide, clopamide, clorexolone, diphenylmethane-4,4′-disulfonamide,disulfamide, ethoxzolamide, furosemide, indapamide, mefruside,methazolamide, piretanide, quinethazone, torasemide, tripamide,xipamide), a uracil (e.g., aminometradine, amisometradine), a potassiumsparing antagonist (e.g., amiloride, triamterene) or a miscellaneousdiuretic such as aminozine, arbutin, chlorazanil, ethacrynic acid,etozolin, hydracarbazine, isosorbide, mannitol, metochalcone,muzolimine, perhexyline, ticmafen and urea. Non-limiting examples of apositive inotropic agent, also known as a cardiotonic, includeacefylline, an acetyldigitoxin, 2-amino-4-picoline, aminone, benfurodilhemisuccinate, bucladesine, cerberosine, camphotamide, convallatoxin,cymarin, denopamine, deslanoside, digitalin, digitalis, digitoxin,digoxin, dobutamine, dopamine, dopexamine, enoximone, erythrophleine,fenalcomine, gitalin, gitoxin, glycocyamine, heptaminol, hydrastinine,ibopamine, a lanatoside, metamivam, milrinone, nerifolin, oleandrin,ouabain, oxyfedrine, prenalterol, proscillaridine, resibufogenin,scillaren, scillarenin, strphanthin, sulmazole, theobromine andxamoterol. In particular aspects, an intropic agent is a cardiacglycoside, a beta-adrenergic agonist or a phosphodiesterase inhibitor.Non-limiting examples of a cardiac glycoside includes digoxin (lanoxin)and digitoxin (crystodigin). Non-limiting examples of a β-adrenergicagonist include albuterol, bambuterol, bitolterol, carbuterol,clenbuterol, clorprenaline, denopamine, dioxethedrine, dobutamine(dobutrex), dopamine (intropin), dopexamine, ephedrine, etafedrine,ethylnorepinephrine, fenoterol, formoterol, hexoprenaline, ibopamine,isoetharine, isoproterenol, mabuterol, metaproterenol, methoxyphenamine,oxyfedrine, pirbuterol, procaterol, protokylol, reproterol, rimiterol,ritodrine, soterenol, terbutaline, tretoquinol, tulobuterol andxamoterol. Non-limiting examples of a phosphodiesterase inhibitorinclude aminone (inocor). Antianginal agents may compriseorganonitrates, calcium channel blockers, beta blockers and combinationsthereof. Non-limiting examples of organonitrates, also known asnitrovasodilators, include nitroglycerin (nitro-bid, nitrostat),isosorbide dinitrate (isordil, sorbitrate) and amyl nitrate (aspirol,vaporole).

2. Disease Cell Cycle Targeting Compounds

Disease cell cycle targeting compounds refers to compounds that targetagents that are upregulated in proliferating cells. Compounds used forthis purpose can be used to measure various parameters in cells, such astumor cell DNA content.

Many of these agents are nucleoside analogues. For example, pyrimidinenucleoside (e.g., 2′-fluoro-2′-deoxy-5-iodo-1-β-D-arabinofuranosyluracil[FIAU], 2′-fluoro-2′-deoxy-5-iodo-1-β-D-ribofuranosyl-uracil [FIRU],2′-fluoro-2′-5-methyl-1-β-D-arabinofuranosyluracil [FMAU],2′-fluoro-2′-deoxy-5-iodovinyl-1-β-D-ribofuranosyluracil [IVFRU]) andacycloguanosine: 9-[(2-hydroxy-1-(hydroxymethyl)ethoxy)methyl]guanine(GCV) and 9-[4-hydroxy-3-(hydroxy-methyl)butyl]guanine (PCV) (Tjuvajevet al., 2002; Gambhir et al., 1998; Gambhir et al., 1999) and other¹⁸F-labeled acycloguanosine analogs, such as8-fluoro-9-[(2-hydroxy-1-(hydroxymethyl)ethoxy)methyl]guanine (FGCV)(Gambhir et al., 1999; Namavari et al., 2000),8-fluoro-9-[4-hydroxy-3-(hydroxymethyl)butyl]guanine (FPCV) (Gambhir etal., 2000; Iyer et al., 2001), 9-[3-fluoro-1-hydroxy-2-propoxymethyl]guanine (FHPG) (Alauddin et al., 1996; Alauddin et al., 1999),and 9-[4-fluoro-3-(hydroxymethyl)butyl]guanine (FHBG) (Alauddin andConti, 1998; Yaghoubi et al., 2001) have been developed as reportersubstrates for imaging wild-type and mutant (Gambhir et al., 2000)HSV1-tk expression. One or ordinary skill in the art would be familiarwith these and other agents that are used for disease cell cycletargeting.

3. Angiogenesis Targeting Ligands

“Angiogenesis targeting ligands” refers to agents that can bind toneovascularization or revascularization of tissue. For example, theneovascularization of tumor cells or revascularization of myocardiumtissue. Agents that are used for this purpose are known to those ofordinary skill in the art for use in performing various measurements,including measurement of the size of a tumor vascular bed andmeasurement of tumor volume. Some of these agents bind to the vascularwall. One of ordinary skill in the art would be familiar with the agentsthat are available for use for this purpose.

Throughout this application, “angiogenesis targeting” refers to the useof an agent to bind to neovascular tissue. Some examples of agents thatare used for this purpose are known to those of ordinary skill in theart for use in performing various tumor measurements, includingmeasurement of the size of a tumor vascular bed, and measurement oftumor volume. Some of these agents bind to the vascular wall. One ofordinary skill in the art would be familiar with the agents that areavailable for use for this purpose. A tumor angiogenesis targetingligand is a ligand that is used for the purpose of tumor angiogenesistargeting as defined above. Examples of angiogenesis targeting ligandsinclude COX-2 inhibitors, anti-EGF receptor ligands, herceptin,angiostatin, C225 and thalidomide. COX-2 inhibitors include, forexample, celecoxib, rofecoxib, etoricoxib and analogs of these agents.

4. Tumor Apoptosis Targeting Ligands

“Tumor apoptosis targeting” refers to use of an agent to bind to a cellthat is undergoing apoptosis or at risk of undergoing apoptosis. Theseagents are generally used to provide an indicator of the extent or riskof apoptosis, or programmed cell death, in a population of cells, suchas a tumor and cardiac tissue. One of ordinary skill in the art would befamiliar with agents that are used for this purpose. A “tumor apoptosistargeting ligand” is a ligand that is capable of performing “tumorapoptosis targeting” as defined in this paragraph. The targeting ligandof the present invention may include TRAIL (TNF-related apoptosisinducing ligand) monoclonal antibody. TRAIL is a member of the tumornecrosis factor ligand family that rapidly induces apoptosis in avariety of transformed cell lines. The targeting ligand of the presentinvention may also comprise a substrate of caspase-3, such as peptide orchelator that includes the 4 amino acid sequence aspartic acid-glutamicacid-valine-aspartic acid. caspase-3 substrate (for example, a peptideor chelator that includes the amino acid sequence aspartic acid-glutamicacid-valine-aspartic acid), and any member of the Bcl family. Examplesof Bcl family members include, for example, Bax, Bcl-xL, Bid, Bad, Bakand Bcl-2. One of ordinary skill in the art would be familiar with theBcl family, and their respective substrates.

Apoptosis suppressors are targets for drug discovery, with the idea ofabrogating their cytoprotective functions and restoring apoptosissensitivity to tumor cells (Reed, 2003).

5. Disease Receptor Targeting Ligands

In “disease receptor targeting,” certain agents are exploited for theirability to bind to certain cellular receptors that are overexpressed indisease states, such as cancer, neurological diseases and cardiovasculardiseases. Examples of such receptors which are targeted include estrogenreceptors, androgen receptors, pituitary receptors, transferrinreceptors and progesterone receptors. Examples of agents that can beapplied in disease-receptor targeting include androgen, estrogen,somatostatin, progesterone, transferrin, luteinizing hormone andluteinizing hormone antibody.

The radiolabeled ligands, such as pentetreotide, octreotide, transferrinand pituitary peptide, bind to cell receptors, some of which areoverexpressed on certain cells. Since these ligands are not immunogenicand are cleared quickly from the plasma, receptor imaging would seem tobe more promising compared to antibody imaging.

The folate receptor is included herein as another example of a diseasereceptor. Folate receptors (FRs) are overexposed on many neoplastic celltypes (e.g., lung, breast, ovarian, cervical, colorectal,nasopharyngeal, renal adenocarcinomas, malignant melanoma andependymomas), but primarily expressed only several normal differentiatedtissues (e.g., choroid plexus, placenta, thyroid and kidney) (Weitman etal., 1992a; Campbell et al., 1991; Weitman et al., 1992b; Holm et al.,1994; Ross et al., 1994; Franklin et al., 1994; Weitman et al., 1994).FRs have been used to deliver folate-conjugated protein toxins,drug/antisense oligonucleotides and liposomes into tumor cellsoverexpressing the folate receptors (Ginobbi et al., 1997; Leamon andLow, 1991; Leamon and Low, 1992; Leamon et al., 1993; Lee and Low,1994). Furthermore, bispecific antibodies that contain anti-FRantibodies linked to anti-T cell receptor antibodies have been used totarget T cells to FR-positive tumor cells and are currently in clinicaltrials for ovarian carcinomas (Canevari et al., 1993; Bolhuis et al.,1992; Patrick et al., 1997; Coney et al., 1994; Kranz et al., 1995).

Examples of folate receptor targeting ligands include folic acid andanalogs of folic acid. Preferred folate receptor targeting ligandsinclude folate, methotrexate and tomudex. Folic acid as well asantifolates such as methotrexate enter into cells via high affinityfolate receptors (glycosylphosphatidylinositol-linked membranefolate-binding protein) in addition to classical reduced-folate carriersystem (Westerhof et al., 1991; Orr et al., 1995; Hsueh and Dolnick,1993).

6. Cardiac Ischemia Markers

In some embodiments, the targeting ligand is a cardiac ischemia marker.A cardiac ischemia marker is a ligand that is relatively selective forischemic cardiac tissue. Non-limiting examples of cardiac ischemiamarkers include interleukin-6, tumor necrosis factor alpha, matrixmetalloproteinase 9, myeloperoxidase, intercellular and vascularadhesion molecules, soluble CD40 ligand, placenta growth factor, highsensitivity C-reactive protein (hs-CRP), ischemia modified albumin(IMA), free fatty acids, and choline.

7. Viability Cardiac Tissue Markers

In some embodiments, the targeting ligand is a viability cardiac tissuemarker. A viability cardiac tissue marker refers to a ligand that isrelatively selective for viable cardiac tissue compared to nonviablecardiac tissue. Non-limiting examples of cardiac viability tissuemarkers include those selected from the group consisting ofphospholipase C, myosin light-chain phosphatase, nitric oxide,prostacyclin, endothelin, thromboxane, L-arginine and L-citrulline.

8. Congestive Heart Failure Markers

In some embodiments, the targeting ligand is a congestive heart failuremarker. A congestive heart failure marker is a ligand that is relativelyselective for cardiac tissue of a heart in congestive heart failurecompared to normal healthy heart tissue. Non-limiting examples ofcongestive heart failure markers include those selected from the groupconsisting of interleukin-1, cardiotrophin-1, insulin-like growthfactor, epidermal growth factor, tyrosine kinase receptor andangiotensin II.

9. Rest/Stress Cardiac Tissue Markers

In some embodiments, the targeting ligand is a rest/stress cardiactissue marker. A rest/stress cardiac tissue marker is a ligand that isrelatively selective for cardiac tissue that is stressed compared tonon-stressed (at rest) cardiac tissue, or vice versa. Non-limitingexamples of rest/stress cardiac tissue markers include those selectedfrom the group consisting of mitogen-activated protein kinase, cyclicadenosine monophosphate, phospholipase C, phosphatidylinositolbisphosphate, isositol trisphosphate, diacylglycerol and tyrosinekinases.

10. Drug Assessment

Certain drug-based ligands can be applied in measuring thepharmacological response of a subject to a drug. A wide range ofparameters can be measured in determining the response of a subject toadministration of a drug. One of ordinary skill in the art would befamiliar with the types of responses that can be measured. Theseresponses depend in part upon various factors, including the particulardrug that is being evaluated, the particular disease or condition forwhich the subject is being treated, and characteristics of the subject.Examples of drug-based ligands include carnitine, puromycin, verapamil,digoxin, prazosin, quinidine, diisopyramide, theophylline, proteaseinhibitors nifedipine, diltiazem, flecaimide, amiodarone, sotalol,adenosine, dopamine dobutamine, inamrinone, milrinone, spironolactone,prazosin, aspirin and warfarin.

11. Antimicrobials

Any antimicrobial is contemplated for inclusion as a targeting ligand.Preferred antimicrobials include ampicillin, amoxicillin, penicillin,clindamycin, gentamycin, kanamycin, neomycin, natamycin, nafcillin,rifampin, tetracycline, vancomycin, bleomycin, doxycyclin, amikacin,netilmicin, streptomycin, tobramycin, loracarbef, ertapenem, imipenem,meropenem, cefadroxil, cefazolin, cephalexin, cefaclor, cefamandole,cefoxitin, cefprozil, cefuroxime, cefixime, cefdinir, cefditoren,cefoperazone, cefotaxime, cefpodoxime, ceftazidime, ceftibuten,ceftizoxime, ceftriaxone, cefepime, teicoplanin, azithromycin,clarithromycin, dirithromycin, erythromycin, roxithromycin,troleandomycin, aztreonam, azlocillin, carbenicillin, cloxacillin,dicloxacillin, flucloxacillin, mezlocillin, piperacillin, ticarcillin,bacitracin, colistin, polymyxin b, ciprofloxacin, enoxacin,gatifloxacin, levofloxacin, lomefloxacin, moxifloxacin, norfloxacin,ofloxacin, trovafloxacin, mafenide, prontosil, sulfacetamide,sulfamethizole, sulfanilamide, sulfasalazine, sulfisoxazole,trimethoprim, trimethoprim-sulfamethoxazole, demeclocycline,minocycline, oxytetracycline, arsphenamine, chloramphenicol, ethambutol,fosfomycin, furazolidone, isoniazid, linezolid, metronidazole,mupirocin, nitrofurantoin, platensimycin, pyrazinamide, quinupristin,dalfopristin, spectinomycin, and telithromycin.

Antifungals include natamycin, rimocidin, filipin, nystatin,amphotericin B, miconazole, ketoconazole, clotrimazole, econazole,bifonazole, butocanazole, finticonazole, isoconazole, oxiconazole,sertaconazole, sulconazole, tioconazole, fluconazole, itraconazole,ravuconazole, posaconazole, voriconazole, terconazole, terbinafine,amorolfine, naftifine, butenafine, anidulafungin, caspofungin,micafungin, ciclopirox, flucytosine, griseofulvin, gentian violet,haloprogin, tolnaftate, undecyclenic acid, amantadine, polymycin,acyclovir and ganciclovir for fungi. One of ordinary skill in the artwould be familiar with the various agents that are considered to beantimicrobials.

12. Agents that Mimic Glucose

Agents that mimic glucose are also contemplated for inclusion astargeting ligands. Such agents can also be considered “glucose analogs”or “glucose derivatives.”

Glucose is utilized by living organisms through the glycolysis pathway.Compounds such as neomycin, kanamycin, gentamycin, amikacin, tobramycin,netilmicin, ribostamycin, sisomicin, micromicin, lividomycin, dibekacin,isepamicin, and astromicin belong to a group called aminoglycosides.

In terms of structure, agents that mimic glucose typically have aglucose ring structure. Exceptions exist, however, such as puromycin,which has a pentose ring structure, but which can still be considered anagent that mimics glucose.

In terms of function, aminoglycosides are used as antibiotics that blockthe glycolysis pathway by their property of being structurally similarto glucose and thus, they are functionally considered as agents thatmimic glucose. When these aminoglycosides are used in imaging studies,there are no detectable pharmacological effects.

The word “mimic”, as defined by the American Heritage Dictionary fourthedition, means “to resemble closely or simulate.” Aminoglycosides arefunctionally utilized through the glycolytic pathway by virtue of theirstructural similarity to glucose and block the glycolysis pathway.Hence, aminoglycosides are considered to mimic or simulate glucose instructural and functional manner.

Non-limiting examples of chemical structures with their PubChem Database(NCBI) identifier CID number are as follows: Amikacin CID 37768;Aminoglycoside CID 191574; Astromicin CID 65345; Deoxy-glucose CID439268; D-glucosamine CID 441477; Dibekacin CID 3021; Gentamicin CID3467; Glucose CID 5793; Isepamicin CID 456297; Kanamycin CID 5460349;Lividomycin CID 72394; Micromicin CID 107677; Neomycin CID 504578;Netilmicin CID 441306; Puromycin CID 439530; Ribostamycin CID 33042;Sisomicin CID 36119; and Tobramycin CID 36294.

References which describe the glycolysis blocking by aminoglycosidesinclude, for example, Tachibana et al., 1976; Borodina et al., 2005;Murakami et al., 1996; Hoelscher et al., 2000; Yang et al., 2004;Michalik et al., 1989; Murakami et al., 1997; Diamond et al., 1978;Hostetler and Hall, 1982; Benveniste and Davies, 1973; Hu, 1998; Yanaiet al., 2006; Myszka et al., 2003; Nakae and Nakae, 1982; Ozmen et al.,2005; and Tod et al., 2000.

Preferred agents that mimic glucose, or sugars, include neomycin,kanamycin, gentamycin, paromycin, amikacin, tobramycin, netilmicin,ribostamycin, sisomicin, micromicin, lividomycin, dibekacin, isepamicin,astromicin, and aminoglycosides glucose and glucosamine.

13. Hypoxia Targeting Ligands

In some embodiments of the present invention, the targeting ligand is atumor hypoxia targeting ligand. For example, tumor cells are moresensitive to conventional radiation in the presence of oxygen than inits absence; even a small percentage of hypoxic cells within a tumorcould limit the response to radiation (Hall, 1988; Bush et al., 1978;Gray et al., 1958). Hypoxic radioresistance has been demonstrated inmany animal tumors but only in few tumor types in humans (Dische, 1991;Gatenby et al., 1988; Nordsmark et al., 1996). The occurrence of hypoxiain human tumors, in most cases, has been inferred from histologyfindings and from animal tumor studies. In vivo demonstration of hypoxiarequires tissue measurements with oxygen electrodes and the invasivenessof these techniques has limited their clinical application.

Misonidazole, an example of a tumor hypoxia targeting ligand, is ahypoxic cell sensitizer, and labeling MISO with different radioisotopes(e.g., ¹⁸F, ¹²³I, ^(99m)Tc) may be useful for differentiating a hypoxicbut metabolically active tumor from a well-oxygenated active tumor byPET or planar scintigraphy. [¹⁸F]Fluoromisonidazole (FMISO) has beenused with PET to evaluate tumors hypoxia. Recent studies have shown thatPET, with its ability to monitor cell oxygen content through [¹⁸F]FMISO,has a high potential to predict tumor response to radiation (Koh et al.,1992; Valk et al., 1992; Martin et al, 1989; Rasey et al., 1989; Raseyet al., 1990; Yang et al., 1995). PET gives higher resolution withoutcollimation, however, the cost of using PET isotopes in a clinicalsetting is prohibitive.

14. Antisense Molecules

Antisense molecules interact with complementary strands of nucleicacids, modifying expression of genes.

Some regions within a double strand of DNA code for genes, which areusually instructions specifying the order of amino acids in a proteinalong with regulatory sequences, splicing sites, noncoding introns andother complicating details. For a cell to use this information, onestrand of the DNA serves as a template for the synthesis of acomplementary strand of RNA. The template DNA strand is called theantisense strand and the RNA is said to be sense (the complement ofantisense). Because the DNA is double-stranded, the strand complementaryto the antisense strand is also called sense and has the same basesequence as the mRNA (though T bases in DNA are substituted with U basesin RNA). For example:

DNA strand 1: sense strand

DNA strand 2: antisense strand (copied to)→RNA strand (sense).

Many forms of antisense have been developed and can be broadlycategorized into enzyme-dependent antisense or steric blockingantisense. Enzyme-dependent antisense includes forms dependent on RNaseH activity to degrade target mRNA, including single-stranded DNA, RNA,and phosphorothioate antisense. Double stranded RNA acts asenzyme-dependent antisense through the RNAi/siRNA pathway, involvingtarget mRNA recognition through sense-antisense strand pairing followedby target mRNA degradation by the RNA-induced silencing complex (RISC).Steric blocking antisense (RNase-H independent antisense) interfereswith gene expression or other mRNA-dependent cellular processes bybinding to a target sequence of mRNA and getting in the way of otherprocesses. Steric blocking antisense includes 2′-O alkyl (usually inchimeras with RNase-H dependent antisense), peptide nucleic acid (PNA),locked nucleic acid (LNA) and Morpholino antisense. Cells can produceantisense RNA molecules naturally, which interact with complementarymRNA molecules and inhibit their expression.

Antisense nucleic acid molecules have been used experimentally to bindto mRNA and prevent expression of specific genes. Antisense therapiesare also in development; the FDA has approved a phosphorothioateantisense oligo, fomivirsen (Vitravene), for human therapeutic use.

15. Imaging Moieties

In certain embodiments of the compositions of the present invention, thetargeting ligand is an imaging moiety. As defined herein, an “imagingmoiety” is a part of a molecule that is a agent or compound that can beadministered to a subject, contacted with a tissue, or applied to a cellfor the purpose of facilitating visualization of particularcharacteristics or aspects of the subject, tissue, or cell through theuse of an imaging modality. Imaging modalities are discussed in greaterdetail below. Any imaging agent known to those of ordinary skill in theart is contemplated as an imaging moiety of the present invention. Thus,for example, in certain embodiments of compositions of the presentinvention, the compositions can be applied in multimodality imagingtechniques. Dual imaging and multimodality imaging are discussed ingreater detail in the specification below.

In certain embodiments, the imaging moiety is a contrast media. Examplesinclude CT contrast media, MRI contrast media, optical contrast media,ultrasound contrast media, or any other contrast media to be used in anyother form of imaging modality known to those of ordinary skill in theart. Examples include diatrizoate (a CT contrast agent), a gadoliniumchelate (an MRI contrast agent) and sodium fluorescein (an opticalcontrast media). Additional examples of contrast media are discussed ingreater detail in the specification below. One of ordinary skill in theart would be familiar with the wide range of types of imaging agentsthat can be employed as imaging moieties in the chelators of the presentinvention.

E. Methods of Synthesis

1. Source of Reagents for the Compositions of the Present Invention

Reagents for preparation of the compositions of the present inventioncan be obtained from any source. A wide range of sources are known tothose of ordinary skill in the art. For example, the reagents can beobtained from commercial sources such as Sigma-Aldrich Chemical Company(Milwaukee, Wis.), from chemical synthesis, or from natural sources. Forexample, one vendor of radionuclides is Cambridge Isotope Laboratories(Andover, Mass.). The reagents may be isolated and purified using anytechnique known to those of ordinary skill in the art, as describedherein. The free unbound metal ions can be removed with, for example,ion-exchange resin or by adding a transchelator (e.g., glucoheptonate,gluconate, glucarate, or acetylacetonate).

2. Use of an Intermediate Product as the Active PharmaceuticalIngredient (API)

Disulfide formation and nucleophilic attack of the anomeric center inthe glucosamine moiety of certain compounds of the present invention canbe problematic. For example, these unwanted reactions may occur at thethiol groups and/or the amino groups in EC-glucosamine (EC-G): these arethe major side reactions that may cause the instability of EC-G.Furthermore, the typically low yield of the deprotection step withNa/NH₃ to get the primary product of EC-G may yield low purity (seeFIGS. 1 and 13). Accordingly, it may be desirable to utilizeintermediates of syntheses of the present invention as activepharmaceutical ingredients (APIs). For example, EC-G analogs such asthose shown below, which are intermediate products in certainpreparations may be used as APIs. These analogs, in certain embodiments,may yield high purity in the scale up process.

3. Purification Procedures and Determinations of Purity

As mentioned above, persons of ordinary skill in the art will befamiliar with methods of purifying compounds of the present invention.As used herein, “purification” refers to any measurable increase inpurity relative to the purity of the material before purification.Purification of every compound of the present invention is generallypossible, including the purification of intermediates as well aspurification of the final products. The purification step is not alwaysincluded in the general methodologies explained below, but one ofordinary skill in the art will understand that compounds can generallybe purified at any step. Examples of purification methods include gelfiltration, size exclusion chromatography (also called gel filtrationchromatography, gel permeation chromatography or molecular exclusion),dialysis, distillation, recrystallization, sublimation, derivatization,electrophoresis, silica gel column chromatography and high-performanceliquid chromatography (HPLC), including normal-phase HPLC andreverse-phase HPLC. In certain embodiments, size exclusionchromatography and/or dialysis are specifically excluded as forms ofpurification of compounds of the present invention. Purification ofcompounds via silica gel column chromatography or HPLC, for example,offer the benefit of yielding desired compounds in very high purity,often higher than when compounds are purified via other methods.Radiochemical purity of compounds of the present invention can also bedetermined. Methods of determining radiochemical purity are well-knownin the art and include chromatographic methods in conjunction withradioactivity detection methods (e.g., autoradiography analyses).Examples of comparisons of purity of compounds made via organic and wetmethodologies and purified by varying methods are provided below.

Methods of determining the purity of compounds are well known to thoseof skill in the art and include, in non-limiting examples,autoradiography, mass spectroscopy, melting point determination, ultraviolet analysis, calorimetric analysis, (HPLC), thin-layerchromatography and nuclear magnetic resonance (NMR) analysis (including,but not limited to, 1H and 13C NMR). In some embodiments, a calorimetricmethod could be used to titrate the purity of a chelator orchelator-targeting ligand conjugate. For instance, generation of athiol-benzyl adduct (that is, a thiol functional group protected by abenzyl group) or the performance of an oxidation reaction by usingiodine could be used to determine the purity of chelator orchelator-targeting ligand conjugate. In one embodiment, the purity of anunknown compound may be determined by comparing it to a compound ofknown purity: this comparison may be in the form of a ratio whosemeasurement describes the purity of the unknown. Software available onvarying instruments (e.g., spectrophotometers, HPLCs, NMRs) can aid oneof skill in the art in making these determinations, as well as othermeans known to those of skill in the art.

The following non-limiting parameters may be used, in certainembodiments, to determine the purity of compounds of the presentinvention:

Column: Primesep100, 4.6×150 mm, 5 μm, ambient temperature

Mobile phase (A): H₂O with 0.025% TFA

Mobile phase (B): acetonitrile with 0.025% TFA

Isocratic run: A/B (50/50) at 1.0 ml/min

Detection: ELSD, SEDEX75, 50C, 4.5 bar

In certain embodiments of the present invention, purification of acompound does not remove all impurities. In some embodiments, suchimpurities can be identified.

4. Obtaining a Chelator

Methods of preparing and obtaining chelators are well known to those ofskill in the art. For example, chelators may be obtained from commercialsources, chemical synthesis, or natural sources.

In one embodiment, the chelator may comprises ethylenedicysteine (EC).The preparation of ethylenedicysteine (EC) is described in U.S. Pat. No.6,692,724. Briefly, EC may be prepared in a two-step synthesis accordingto the previously described methods (Ratner and Clarke, 1937; Blondeauet al., 1967; each incorporated herein by reference). The precursor,L-thiazolidine-4-carboxylic acid, was synthesized and then EC was thenprepared. It is often also important to include an antioxidant in thecomposition to prevent oxidation of the ethylenedicysteine. Thepreferred antioxidant for use in conjunction with the present inventionis vitamin C (ascorbic acid). However, it is contemplated that otherantioxidants, such as tocopherol, pyridoxine, thiamine, or rutin mayalso be useful.

Chelators may also comprise amino acids joined together by spacers. Sucha spacer may comprise, as described above, an alkyl spacer such asethylene.

Amide bonds may also join one or more amino acids together to form achelator. Examples of synthetic methods for the preparation of suchchelators include solid-phase synthesis and solution-phase synthesis.Such methods are described, for example, in Bodansky, 1993 and Grant,1992.

5. Organic Synthesis of Chelator-Targeting Ligand Conjugates

In a preferred embodiment, the present invention further provides amethod of organically synthesizing chelator-targeting ligand conjugates.The method includes obtaining, for example, a chelator such asethylenedicysteine (EC) as described above and admixing the EC with athiol protecting group in an organic medium in order to protect bothfree thiols, resulting in an S—S′-bis-protected-EC, which is thenadmixed with an amino protecting group in an organic/aqueous medium inorder to protect both free amines, resulting in anS—S′-bis-protected-N,N′-bis-protected-EC. Thiol groups are more reactivethan nitrogen groups; thus, thiol groups are typically protected first.As described above, persons of skill in the art will be familiar withthe proper ordering of the installation of protecting groups dependingon the types of functional groups present on the chelator. Thisprotected EC is then conjugated to a targeting ligand of any typedescribed herein via any mode of conjugation described herein followedby removal of the thiol and amino protecting groups, which results in achelator-targeting ligand conjugate.

In certain embodiments, conjugation between a chelator and a targetingligand takes place in one step. In particular embodiments, theconjugation comprises a covalent attachment of a chelator to a targetingligand, wherein the covalent attachment occurs in one step. Asmentioned, such one-step procedures are preferable as they minimizetime, reagents, waste and loss of product.

Chelator-targeting ligand conjugates synthesized by this method may nextbe chelated to a metal ion of any type described herein. Such methods ofchelation are well known to those of ordinary skill in the art and aredescribed herein. Examples of methods of chelation of metal ions tochelator-targeting ligand conjugates are described, for example, in U.S.Pat. No. 6,692,724. Methods described herein where a metal ion ischelated to a chelator may also serve as examples of how to chelate ametal ion to a chelator-targeting ligand conjugate.

Benefits of synthesizing chelator-targeting ligand conjugates viamethods of the present invention using organic synthesis include, forexample, obtaining conjugates of high purity relative to conjugatesobtained via aqueous synthesis, and the efficient synthesis andpurification of small-molecule compounds (e.g., 1000 g/mol or less).These benefits allow for conjugates that can be utilized in imaging,diagnostic, and/or therapeutic experiments and/or clinical trials.

6. Organic Synthesis of Chelator-Targeting Ligand Conjugates Chelated toa Metal Ion

In another preferred embodiment, the present invention further providesa method of organically synthesizing chelator-targeting ligandconjugates chelated to a metal ion for imaging, diagnostic, ortherapeutic use. The method includes, for example, first obtaining achelator, such as EC. EC may then admixed with a metal ion, which may bea radionuclide or any other metal ion as described herein, in an organicmedium in order to chelate to the EC via an N₂S₂ chelate. See, e.g.,FIG. 2. Other methods of chelation are described herein (e.g., chelatesof any combination of O, N and S) and chelation may occur by any methoddescribed herein. In non-limiting examples, metals such as technetium,indium, rhenium, gallium, copper, holmium, platinum, gadolinium,lutetium, yttrium, cobalt, calcium and arsenic can be chelated with achelator such as EC. The EC chelated to a metal ion (“chelated EC”) isthen admixed with a targeting ligand, optionally protected with one ormore protecting groups, in the presence of an organic medium in order togenerate a chelator-targeting ligand conjugate chelated to a metal ion.The mode of conjugation may be via any mode described herein and maytake place in one step or in more than one step.

Benefits of synthesizing metal ion-labeled chelator-targeting ligandconjugates via methods of the present invention using organic synthesisinclude, for example, obtaining conjugates of high purity relative toconjugates obtained via aqueous synthesis, and the efficient synthesisand purification of small-molecule compounds (e.g., 1000 g/mol or less).These benefits allow for conjugates that can be utilized in imaging,diagnostic, and/or therapeutic experiments and/or clinical trials.

7. Aqueous Synthesis of Chelator-Targeting Ligand Conjugates

The present invention further provides a method of synthesizingchelator-targeting ligand conjugates in an aqueous medium.Chelator-targeting ligand conjugates were prepared, in general, as ameans of comparing the relative purity of such or similar products whensynthesized in organic mediums. The method includes, for example, firstobtaining a chelator, such as EC. EC is then dissolved in a basicaqueous solution and coupling agents of any type described herein areadded. The targeting ligand is then added to this solution to generatethe chelator-targeting ligand conjugate.

8. Aqueous Synthesis of Chelator-Targeting Ligand Conjugates Chelated toa Metal Ion

The present invention further provides a method of synthesizing, in anaqueous medium, chelator-targeting ligand conjugates chelated to a metalion. Like the aqueous synthesis mentioned above, chelator-targetingligands conjugates chelated to a metal ion were prepared as a means ofcomparing the relative purity of such or similar products whensynthesized in organic mediums. The method commences, in one embodiment,with obtaining a chelator chelated to a metal ion as described above(“Organic Synthesis of Chelator-Targeting Ligand Conjugates Chelated toa Metal Ion”). This chelator chelated to a metal ion may be, forexample, chelated EC as described above. Chelation may occur by anymethod described herein. Chelated EC may be dissolved in a basic aqueoussolution and coupling agents, as described herein, are added along witha targeting ligand of any type described herein in order to generate achelator-targeting ligand conjugate chelated to a metal ion.

9. Conjugation of a Chelator with a Targeting Ligand

The present invention contemplates methods for conjugating a targetingligand to a chelator (optionally chelated to a metal ion). The targetingligand may be of any type described herein. One of ordinary skill in theart will be familiar with the means of conjugating targeting ligands tovarious functional groups. Most commonly, as between the chelator andthe targeting ligand, one acts as the nucleophile and one acts as theelectrophile such that conjugation takes place via a covalent bond.Non-limiting examples of such covalent bonds include an amide bond, anester bond, a thioester bond and a carbon-carbon bond. In preferredembodiments, the conjugation takes place via an amide or ester bond. Insome embodiments, the conjugation takes place at one or more functionalgroups of the chelator selected from the group consisting of carboxylicacid, amine and thiol. When acting as electrophiles, chelators andtargeting ligands may comprise functional groups such as halogens andsulfonyls which act as leaving groups during conjugation. Targetingligands may also comprise nucleophilic groups, such as —NH₂, which mayparticipate in conjugation with an electrophilic chelator.

Coupling agents, as used herein, are reagents used to facilitate thecoupling of a chelator to a targeting ligand. Such agents are well knownto those of ordinary skill in the art and may be employed in certainembodiments of methods of the present invention. Examples of couplingagents include, but are not limited to, sulfo-N-hydroxysuccinimide(sulfo-NHS), dimethylaminopyridine (DMAP),diazabicyclo[5.4.0]undec-7-ene (DBU),1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC) anddicyclohexylcarbodiimide (DCC). Other carbodiimides are also envisionedas coupling agents. Coupling agents are discussed, for example, inBodansky, 1993 and Grant, 1992. These coupling agents may be used singlyor in combination with each other or other agents to facilitateconjugation. Once the targeting ligand is conjugated using a couplingagent, urea is typically formed. The urea by-product may be removed byfiltration. The conjugated product may then be purified by, for example,silica gel column chromatography or HPLC.

In general, the ligands for use in conjunction with the presentinvention will possess functional groups that are able to conjugate toone or more functional groups of a chelator, such as EC. For example, atargeting ligand may possess a halogenated position that will react witha free amine of a chelator to form the conjugate. If functional groupsare not available, or if an optimal functional group is not available, adesired ligand may still be conjugated to a chelator, such as EC, byadding a linker, such as ethylenediamine, amino propanol,diethylenetriamine, aspartic acid, polyaspartic acid, glutamic acid,polyglutamic acid, cysteine, glycine or lysine. For example, U.S. Pat.No. 6,737,247 discloses several linkers which may be used with thepresent invention and is hereby incorporated by reference in itsentirety without disclaimer. U.S. Pat. No. 5,605,672 discloses several“preferred backbones” which may be used as linkers in the presentinvention and is hereby incorporated by reference in its entirety. Incertain embodiments, the chelator may be conjugated to a linker, and thelinker is conjugated to the targeting ligand. In other embodiments morethan one linker may be used; for example, a chelator may be conjugatedto a linker, and the linker is conjugated to a second linker, whereinthe second linker is conjugated to the targeting ligand. In certainembodiments, two, three, four, or more linkers that are conjugatedtogether may be used to conjugate a chelator and targeting ligand.However, it is generally preferable to only use a single linker toconjugate a chelator and a targeting ligand.

Some chelators, such as EC, are water soluble. In some embodiments, thechelator-targeting ligand conjugate chelated to a metal ion of theinvention is water soluble. Many of the targeting ligands used inconjunction with the present invention will be water soluble, or willform a water soluble compound when conjugated to the chelator. If thetargeting ligand is not water soluble, however, a linker which willincrease the solubility of the ligand may be used. Linkers may attachto, for example, an aliphatic or aromatic alcohol, amine, peptide or toa carboxylic acid. Linkers may be, for example, either poly amino acids(peptides) or amino acids such as glutamic acid, aspartic acid orlysine. Table 2 illustrates preferred linkers for specific drugfunctional groups.

Benefits of synthesizing chelator-targeting ligand conjugates optionallychelated to one or more valent metal ions via methods of the presentinvention using organic synthesis include, for example, obtainingconjugates of high purity relative to conjugates obtained via aqueoussynthesis, and the efficient synthesis and purification ofsmall-molecule compounds (e.g., 1000 g/mol or less). These benefitsallow for conjugates that can be utilized in imaging, diagnostic, and/ortherapeutic experiments and/or clinical trials.

TABLE 2 Linkers Drug Functional Group Linker Example Aliphatic orEC-poly(glutamic acid) estradiol, topotecan, phenolic-OH (MW 750-15,000)or EC paclitaxel, raloxifen poly(aspartic acid) (MW etoposide2000-15,000) or bromo ethylacetate or EC-glutamic acid or EC-asparticacid. Aliphatic or EC-poly(glutamic acid) doxorubicin, aromatic-NH₂ (MW750-15,000) or EC- mitomycin C, or peptide poly(aspartic acid) (MWendostatin, annexin V, 2000-15,000) or EC- LHRH, octreotide, glutamicacid (mono- or VIP diester) or EC-aspartic acid. Carboxylic acid orEthylene diamine, lysine methotrexate, folic peptide acid

10. Chelation of a Metal Ion

The present invention further contemplates methods for the chelation(also called coordination) of one or more metal ions to a chelator or achelator-targeting ligand conjugate. Such chelation steps may take placein organic media. In other embodiments, chelation takes place in aqueousmedia. In certain embodiments, the chelator and the targeting ligand mayeach contribute to the chelation of the metal ion. In preferredembodiments, the metal ion is chelated only to the chelator. Thechelated metal ion may be bound via, for example, an ionic bond, acovalent bond, or a coordinate covalent bond (also called a dativebond). Methods of such coordination are well known to those of ordinaryskill in the art. In one embodiment, coordination may occur by admixinga metal ion into a solution containing a chelator. In anotherembodiment, coordination may occur by admixing a metal ion into asolution containing a chelator-targeting ligand conjugate. In oneembodiment, chelation occurs to the chelator, with or without atargeting ligand, via an N₂S₂ chelate formed by the chelator, such asethylenedicysteine (EC). The chelator and the targeting ligand may eachbe protected by one or more protecting groups before or after chelationwith the metal ion.

Chelation may occur at any atom or functional group of a chelator ortargeting ligand that is available for chelation. The chelation mayoccur, for example, at one or more N, S, O or P atoms. Non-limitingexamples of chelation groups include NS₂, N₂S, S₄, N₂S₂, N₃S and NS₃,and O₄. In preferred embodiments, a metal ion is chelated to three orfour atoms. In some embodiments, the chelation occurs among one or morethiol, amine or carboxylic acid functional groups. The chelation, inparticular embodiments, may be to a carboxyl moiety of glutamate,aspartate, an analog of glutamate, or an analog of aspartate. Theseembodiments may include multiple metal ions chelated to poly(glutamate)or poly(aspartate) chelators. In some embodiments, chelation of themetal ion is to a targeting ligand, such as to carboxyl groups of atissue-specific ligand. In preferred embodiments, the chelation isbetween one or more thiol groups and one or more amine groups of thechelator.

In some non-limiting examples, the metal ion may be technetium, indium,rhenium, gallium, copper, holmium, platinum, gadolinium, lutetium,yttrium, cobalt, calcium, arsenic, or any isotope thereof. Any metal iondescribed herein may be chelated to a compound of the present invention.

11. Reducing Agents

For purposes of the present invention, when the metal ion is technetiumit is preferred that the Tc be in the +4 oxidation state. The preferredreducing agent for use this purpose is stannous ion in the form ofstannous chloride (SnCl₂) to reduce the Tc to its +4 oxidation state.However, it is contemplated that other reducing agents, such asdithionate ion or ferrous ion may be useful in conjunction with thepresent invention. It is also contemplated that the reducing agent maybe a solid phase reducing agent. The amount of reducing agent can beimportant as it is necessary to avoid the formation of a colloid. It ispreferable, for example, to use from about 10 to about 100 μg SnCl₂ perabout 100 to about 300 mCi of Tc pertechnetate. The most preferredamount is about 0.1 mg SnCl₂ per about 200 mCi of Tc pertechnetate andabout 2 mL saline. This typically produces enough Tc-EC-targeting ligandconjugate for use in 5 patients.

F. Examples of Imaging Modalities

1. Gamma Camera Imaging

A variety of nuclear medicine techniques for imaging are known to thoseof ordinary skill in the art. Any of these techniques can be applied inthe context of the imaging methods of the present invention to measure asignal from the reporter. For example, gamma camera imaging iscontemplated as a method of imaging that can be utilized for measuring asignal derived from the reporter. One of ordinary skill in the art wouldbe familiar with techniques for application of gamma camera imaging(see, e.g., Kundra et al, 2002, herein specifically incorporated byreference). In one embodiment, measuring a signal can involve use ofgamma-camera imaging of a 111-In-octreotide-SSRT2A reporter system.

2. PET and SPECT

Radionuclide imaging modalities (positron emission tomography (PET);single photon emission computed tomography (SPECT)) are diagnosticcross-sectional imaging techniques that map the location andconcentration of radionuclide-labeled radiotracers. Although CT and MRIprovide considerable anatomic information about the location and theextent of tumors, these imaging modalities cannot adequatelydifferentiate invasive lesions from edema, radiation necrosis, gradingor gliosis. PET and SPECT can be used to localize and characterizetumors by measuring metabolic activity.

PET and SPECT provide information pertaining to information at thecellular level, such as cellular viability. In PET, a patient ingests oris injected with a slightly radioactive substance that emits positrons,which can be monitored as the substance moves through the body. In onecommon application, for instance, patients are given glucose withpositron emitters attached, and their brains are monitored as theyperform various tasks. Since the brain uses glucose as it works, a PETimage shows where brain activity is high.

Closely related to PET is single-photon emission computed tomography, orSPECT. The major difference between the two is that instead of apositron-emitting substance, SPECT uses a radioactive tracer that emitslow-energy photons. SPECT is valuable for diagnosing coronary arterydisease, and already some 2.5 million SPECT heart studies are done inthe United States each year.

PET radiopharmaceuticals for imaging are commonly labeled withpositron-emitters such as ¹¹C, ¹³N, ¹⁵O, ¹⁸F, ⁸²Rb, ⁶²Cu, and ⁶⁸Ga.SPECT radiopharmaceuticals are commonly labeled with positron emitterssuch as ^(99m)Tc, ²⁰¹Tl, and ⁶⁷Ga. Regarding brain imaging, PET andSPECT radiopharmaceuticals are classified according toblood-brain-barrier permeability (BBB), cerebral perfusion andmetabolism receptor-binding, and antigen-antibody binding (Saha et al.,1994). The blood-brain-barrier SPECT agents, such as ^(99m)TcO4-DTPA,²⁰¹ Tl, and [⁶⁷ Ga]citrate are excluded by normal brain cells, but enterinto tumor cells because of altered BBB. SPECT perfusion agents such as[¹²³I]IMP, [^(99m)Tc]HMPAO, [^(99m)Tc]ECD are lipophilic agents, andtherefore diffuse into the normal brain. Important receptor-bindingSPECT radiopharmaceuticals include [¹²³I]QNE, [¹²³I]IBZM, and[¹²³I]iomazenil. These tracers bind to specific receptors, and are ofimportance in the evaluation of receptor-related diseases.

3. Computerized Tomography (CT)

Computerized tomography (CT) is contemplated as an imaging modality inthe context of the present invention. By taking a series of X-rays,sometimes more than a thousand, from various angles and then combiningthem with a computer, CT made it possible to build up athree-dimensional image of any part of the body. A computer isprogrammed to display two-dimensional slices from any angle and at anydepth.

In CT, intravenous injection of a radiopaque contrast agent can assistin the identification and delineation of soft tissue masses when initialCT scans are not diagnostic. Similarly, contrast agents aid in assessingthe vascularity of a soft tissue or bone lesion. For example, the use ofcontrast agents may aid the delineation of the relationship of a tumorand adjacent vascular structures.

CT contrast agents include, for example, iodinated contrast media.Examples of these agents include iothalamate, iohexyl, diatrizoate,iopamidol, ethiodol and iopanoate. Gadolinium agents have also beenreported to be of use as a CT contrast agent (see, e.g., Henson et al.,2004). For example, gadopentate agents has been used as a CT contrastagent (discussed in Strunk and Schild, 2004).

4. Magnetic Resonance Imaging (MRI)

Magnetic resonance imaging (MRI) is an imaging modality that is newerthan CT that uses a high-strength magnet and radio-frequency signals toproduce images. The most abundant molecular species in biologicaltissues is water. It is the quantum mechanical “spin” of the waterproton nuclei that ultimately gives rise to the signal in imagingexperiments. In MRI, the sample to be imaged is placed in a strongstatic magnetic field (1-12 Tesla) and the spins are excited with apulse of radio frequency (RF) radiation to produce a net magnetizationin the sample. Various magnetic field gradients and other RF pulses thenact on the spins to code spatial information into the recorded signals.By collecting and analyzing these signals, it is possible to compute athree-dimensional image which, like a CT image, is normally displayed intwo-dimensional slices.

Contrast agents used in MR imaging differ from those used in otherimaging techniques. Their purpose is to aid in distinguishing betweentissue components with identical signal characteristics and to shortenthe relaxation times (which will produce a stronger signal onT1-weighted spin-echo MR images and a less intense signal on T2-weightedimages). Examples of MRI contrast agents include gadolinium chelates,manganese chelates, chromium chelates, and iron particles.

Both CT and MRI provide anatomical information that aid indistinguishing tissue boundaries and vascular structure. Compared to CT,the disadvantages of MRI include lower patient tolerance,contraindications in pacemakers and certain other implanted metallicdevices, and artifacts related to multiple causes, not the least ofwhich is motion (Alberico et al, 2004). CT, on the other hand, is fast,well tolerated, and readily available but has lower contrast resolutionthan MRI and requires iodinated contrast and ionizing radiation(Alberico et al., 2004). A disadvantage of both CT and MRI is thatneither imaging modality provides functional information at the cellularlevel. For example, neither modality provides information regardingcellular viability.

5. Optical Imaging

Optical imaging is another imaging modality that has gained widespreadacceptance in particular areas of medicine. Examples include opticallabelling of cellular components, and angiography such as fluoresceinangiography and indocyanine green angiography. Examples of opticalimaging agents include, for example, fluorescein, a fluoresceinderivative, indocyanine green, Oregon green, a derivative of Oregongreen derivative, rhodamine green, a derivative of rhodamine green, aneosin, an erythrosin, Texas red, a derivative of Texas red, malachitegreen, nanogold sulfosuccinimidyl ester, cascade blue, a coumarinderivative, a naphthalene, a pyridyloxazole derivative, cascade yellowdye, or dapoxyl dye.

6. Ultrasound

Another biomedical imaging modality that has gained widespreadacceptance is ultrasound. Ultrasound imaging has been used noninvasivelyto provide realtime cross-sectional and even three-dimensional images ofsoft tissue structures and blood flow information in the body.High-frequency sound waves and a computer to create images of bloodvessels, tissues and organs.

Ultrasound imaging of blood flow can be limited by a number of factorssuch as size and depth of the blood vessel. Ultrasonic contrast agents,a relatively recent development, include perfluorine and perfluorineanalogs, which are designed to overcome these limitations by helping toenhance grey-scale images and Doppler signals.

7. Procedure for Dual Imaging

Certain embodiments of the present invention pertain to methods ofimaging a site within a subject using two imaging modalities thatinvolve measuring a first signal and a second signal from the imagingmoiety-chelator-metal ion complex. The first signal is derived from themetal ion and the second signal is derived from the imaging moiety. Asset forth above, any imaging modality known to those of ordinary skillin the art can be applied in these embodiments of the present imagingmethods.

The imaging modalities are performed at any time during or afteradministration of the composition comprising the diagnosticallyeffective amount of the composition of the present invention. Forexample, the imaging studies may be performed during administration ofthe dual imaging composition of the present invention, or at any timethereafter. In some embodiments, the first imaging modality is performedbeginning concurrently with the administration of the dual imagingagent, or about 1 sec, 1 hour, 1 day, or any longer period of timefollowing administration of the dual imaging agent, or at any time inbetween any of these stated times.

The second imaging modality may be performed concurrently with the firstimaging modality, or at any time following the first imaging modality.For example, the second imaging modality may be performed about 1 sec,about 1 hour, about 1 day, or any longer period of time followingcompletion of the first imaging modality, or at any time in between anyof these stated times. In certain embodiments of the present invention,the first and second imaging modalities are performed concurrently suchthat they begin at the same time following administration of the agent.One of ordinary skill in the art would be familiar with performance ofthe various imaging modalities contemplated by the present invention.

In some embodiments of the present methods of dual imaging, the sameimaging device is used to perform a first imaging modality and a secondimaging modality. In other embodiments, a different imaging device isused to perform the second imaging modality. One of ordinary skill inthe art would be familiar with the imaging devices that are availablefor performance of a first imaging modality and a second imagingmodality, and the skilled artisan would be familiar with use of thesedevices to generate images.

G. Radiolabeled Agents

As set forth above, certain embodiments of the compositions of thepresent invention include a metal ion chelated to a chelator as setforth above. In some embodiments, the metal ion is a radionuclide.Radiolabeled agents, compounds, and compositions provided by the presentinvention are provided having a suitable amount of radioactivity. Forexample, in forming ^(99m)Tc radioactive complexes, it is generallypreferred to form radioactive complexes in solutions containingradioactivity at concentrations of from about 0.01 millicurie (mCi) toabout 300 mCi per mL.

Radiolabeled imaging agents provided by the present invention can beused for visualizing sites in a mammalian body. In accordance with thisinvention, the imaging agents are administered by any method known tothose of ordinary skill in the art. For example, administration may bein a single unit injectable dose. Any of the common carriers known tothose with skill in the art, such as sterile saline solution or plasma,may be utilized after radiolabeling for preparing the compounds of thepresent invention for injection. Generally, a unit dose to beadministered has a radioactivity of about 0.01 mCi to about 300 mCi,preferably 10 mCi to about 200 mCi. The solution to be injected at unitdosage is from about 0.01 mL to about 10 mL.

After intravenous administration of a diagnostically effective amount ofa composition of the present invention, imaging can be performed.Imaging of a site within a subject, such as an organ or tumor can takeplace, if desired, in hours or even longer, after the radiolabeledreagent is introduced into a patient. In most instances, a sufficientamount of the administered dose will accumulate in the area to be imagedwithin about 0.1 of an hour. As set forth above, imaging may beperformed using any method known to those of ordinary skill in the art.Examples include PET, SPECT, and gamma scintigraphy. In gammascintigraphy, the radiolabel is a gamma-radiation emitting radionuclideand the radiotracer is located using a gamma-radiation detecting camera.The imaged site is detectable because the radiotracer is chosen eitherto localize at a pathological site (termed positive contrast) or,alternatively, the radiotracer is chosen specifically not to localize atsuch pathological sites (termed negative contrast).

H. Kits

Certain embodiments of the present invention are generally concernedwith kits for preparing an imaging or diagnostic agent. For example, insome embodiments the kit includes one or more sealed containers thatcontain a predetermined quantity of a chelator-targeting ligandconjugate. In some embodiments, the kit further includes a sealedcontainer containing a metal ion. For example, the metal ion may be aradionuclide or a cold metal ion.

A kit of the present invention may include a sealed vial containing apredetermined quantity of a chelator of the present invention and asufficient amount of reducing agent to label the compound with a metalion. In some embodiments of the present invention, the kit includes ametal ion that is a radionuclide. In certain further embodiments, theradionuclide is ^(99m)Tc. In further embodiments of the presentinvention, the chelator is conjugated to a targeting ligand that can beany of those targeting ligands discussed elsewhere in this application.

The kit may also contain conventional pharmaceutical adjunct materialssuch as, for example, pharmaceutically acceptable salts to adjust theosmotic pressure, buffers, preservatives and the like.

In certain embodiments, an antioxidant is included in the composition toprevent oxidation of the chelator moiety. In certain embodiments, theantioxidant is vitamin C (ascorbic acid). However, it is contemplatedthat any other antioxidant known to those of ordinary skill in the art,such as tocopherol, pyridoxine, thiamine, or rutin, may also be used.The components of the kit may be in liquid, frozen, or dry form. In apreferred embodiment, kit components are provided in lyophilized form.

The cold (that is, non-radioactivity containing) instant kit isconsidered to be a commercial product. The cold instant kit could servea radiodiagnostic purpose by adding pertechnetate to vial with API andbulking agents (agents which have not been tested yet). The technologyis known as the “shake and shoot” method to those of skill in the art.The preparation time of radiopharmaceuticals would be less than 15 min.The same kit could also encompass chelators or chelator-targeting ligandconjugates that could be chelated with different metals for differentimaging applications. For instance, copper-61 (3.3 hrs half life) forPET; gadolinium for MRI. The cold kit itself could be used for prodrugpurposes to treat disease. For example, the kit could be applied intissue-specific targeted imaging and therapy.

I. Hyperproliferative Disease

Certain aspects of the present invention pertain to compositions whereina therapeutic moiety is conjugated to a chelator of the presentinvention. When a metal ion is chelated to a chelator or to both achelator and its conjugated targeting ligand, the composition of thepresent invention may, in certain embodiments, be useful in dual imagingand therapy. In certain particular embodiments, the therapeutic moietyis a moiety that is an agent known or suspected to be of benefit in thetreatment or prevention of hyperproliferative disease in a subject. Thesubject may be an animal, such as a mammal. In certain particularembodiments, the subject is a human.

In other embodiments of the present invention, the metal ion is atherapeutic metal ion (e.g., Re-188, Re-187, Re-186, Ho-166, Y-90,Sr-89, and Sm-153), and the chelator-metal ion chelate is an agent thatis a therapeutic agent (rather than an imaging agent) that can beapplied in the treatment or prevention of a hyperproliferative disease.

A hyperproliferative disease is herein defined as any disease associatedwith abnormal cell growth or abnormal cell turnover. For example, thehyperproliferative disease may be cancer. The term “cancer” as usedherein is defined as an uncontrolled and progressive growth of cells ina tissue. A skilled artisan is aware other synonymous terms exist, suchas neoplasm or malignancy or tumor. Any type of cancer is contemplatedfor treatment by the methods of the present invention. For example, thecancer may be breast cancer, lung cancer, ovarian cancer, brain cancer,liver cancer, cervical cancer, colon cancer, renal cancer, skin cancer,head and neck cancer, bone cancer, esophageal cancer, bladder cancer,uterine cancer, stomach cancer, pancreatic cancer, testicular cancer,lymphoma, or leukemia. In other embodiments of the present invention,the cancer is metastatic cancer.

J. Dual Chemotherapy and Radiation Therapy (“Radiochemotherapy”)

In certain embodiments of the present invention, the compositions of thepresent invention are suitable for dual chemotherapy and radiationtherapy (radiochemotherapy). For example, the chelator as set forthherein may be chelated to a metal ion that is a therapeutic metal ion,as well as a targeting ligand that is a therapeutic moiety (such as ananti-cancer moiety). As another example, a therapeutic metal ion may bechelated to both a chelator and its targeting ligand conjugate.

For example, the metal ion may be a beta-emitter. As herein defined, abeta emitter is any agent that emits beta energy of any range. Examplesof beta emitters include Re-188, Re-187, Re-186, Ho-166, Y-90, andSn-153. One of ordinary skill in the art would be familiar with theseagents for use in the treatment of hyperproliferative disease, such ascancer.

One of ordinary skill in the art would be familiar with the design ofchemotherapeutic protocols and radiation therapy protocols that canapplied in the administration of the compounds of the present invention.As set forth below, these agents may be used in combination with othertherapeutic modalities directed at treatment of a hyperproliferativedisease, such as cancer. Furthermore, one of ordinary skill in the artwould be familiar with selecting an appropriate dose for administrationto the subject. The protocol may involve a single dose, or multipledoses. The patient would be monitored for toxicity and response totreatment using protocols familiar to those of ordinary skill in theart.

K. Pharmaceutical Preparations

Pharmaceutical compositions of the present invention comprise atherapeutically or diagnostically effective amount of a composition ofthe present invention. The phrases “pharmaceutical or pharmacologicallyacceptable” or “therapeutically effective” or “diagnostically effective”refers to molecular entities and compositions that do not produce anadverse, allergic or other untoward reaction when administered to ananimal, such as, for example, a human, as appropriate. The preparationof therapeutically effective or diagnostically effective compositionswill be known to those of skill in the art in light of the presentdisclosure, as exemplified by Remington's Pharmaceutical Sciences, 18thEd. Mack Printing Company, 1990, incorporated herein by reference.Moreover, for animal (e.g., human) administration, it will be understoodthat preparations should meet sterility, pyrogenicity, general safetyand purity standards as required by the FDA Office of BiologicalStandards.

As used herein, “a composition comprising a therapeutically effectiveamount” or “a composition comprising a diagnostically effective amount”includes any and all solvents, dispersion media, coatings, surfactants,antioxidants, preservatives (e.g., antibacterial agents, antifungalagents), isotonic agents, absorption delaying agents, salts,preservatives, drugs, drug stabilizers, gels, binders, excipients,disintegration agents, lubricants, sweetening agents, flavoring agents,dyes, such like materials and combinations thereof, as would be known toone of ordinary skill in the art. Except insofar as any conventionalcarrier is incompatible with the active ingredient, its use in thepresent compositions is contemplated.

The compositions of the present invention may comprise different typesof carriers depending on whether it is to be administered in solid,liquid or aerosol form, and whether it need to be sterile for suchroutes of administration as injection. The compositions of the presentinvention can be administered intravenously, intradermally,intraarterially, intraperitoneally, intralesionally, intracranially,intraarticularly, intraprostaticaly, intrapleurally, intratracheally,intranasally, intravitreally, intravaginally, intrarectally, topically,intratumorally, intramuscularly, intraperitoneally, subcutaneously,subconjunctival, intravesicularlly, mucosally, intrapericardially,intraumbilically, intraocularally, orally, topically, locally,injection, infusion, continuous infusion, localized perfusion bathingtarget cells directly, via a catheter, via a lavage, in lipidcompositions (e.g., liposomes), or by other method or any combination ofthe forgoing as would be known to one of ordinary skill in the art.

The actual required amount of a composition of the present inventionadministered to a patient can be determined by physical andphysiological factors such as body weight, severity of condition, thetissue to be imaged, the type of disease being treated, previous orconcurrent imaging or therapeutic interventions, idiopathy of thepatient, and on the route of administration. The practitionerresponsible for administration will, in any event, determine theconcentration of active ingredient(s) in a composition and appropriatedose(s) for the individual subject.

In certain embodiments, pharmaceutical compositions may comprise, forexample, at least about 0.1% of the chelator-metal ion chelate. In otherembodiments, the an active compound may comprise between about 2% toabout 75% of the weight of the unit, or between about 25% to about 60%,for example, and any range derivable therein. In other non-limitingexamples, a dose may also comprise from about 0.1 mg/kg/body weight toabout 1000 mg/kg/body weight or any amount within this range, or anyamount greater than 1000 mg/kg/body weight per administration.

In any case, the composition may comprise various antioxidants to retardoxidation of one or more component. Additionally, the prevention of theaction of microorganisms can be brought about by preservatives such asvarious antibacterial and antifungal agents, including, but not limitedto parabens (e.g., methylparabens, propylparabens), chlorobutanol,phenol, sorbic acid, thimerosal or combinations thereof.

The compositions of the present invention may be formulated in a freebase, neutral or salt form. Pharmaceutically acceptable salts includethe salts formed with the free carboxyl groups derived from inorganicbases such as for example, sodium, potassium, ammonium, calcium orferric hydroxides; or such organic bases as isopropylamine,trimethylamine, histidine or procaine.

In embodiments where the composition is in a liquid form, a carrier canbe a solvent or dispersion medium comprising, but not limited to, water,ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethyleneglycol, etc.), lipids (e.g., triglycerides, vegetable oils, liposomes)and combinations thereof. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin; by the maintenanceof the required particle size by dispersion in carriers such as, forexample liquid polyol or lipids; by the use of surfactants such as, forexample hydroxypropylcellulose; or combinations thereof such methods. Inmany cases, it will be preferable to include isotonic agents, such as,for example, sugars, sodium chloride or combinations thereof.

Sterile injectable solutions may be prepared using techniques such asfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and/or the otheringredients. In the case of sterile powders for the preparation ofsterile injectable solutions, suspensions or emulsion, the preferredmethods of preparation are vacuum-drying or freeze-drying techniqueswhich yield a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered liquid mediumthereof. The liquid medium should be suitably buffered if necessary andthe liquid diluent first rendered isotonic prior to injection withsufficient saline or glucose. The preparation of highly concentratedcompositions for direct injection is also contemplated, where the use ofDMSO (dimethylsulfoxide) as solvent is envisioned to result in extremelyrapid penetration, delivering high concentrations of the active agentsto a small area.

The composition must be stable under the conditions of manufacture andstorage, and preserved against the contaminating action ofmicroorganisms, such as bacteria and fungi. It will be appreciated thatendotoxin contamination should be kept minimally at a safe level, forexample, less that 0.5 ng/mg protein.

In particular embodiments, prolonged absorption of an injectablecomposition can be brought about by the use in the compositions ofagents delaying absorption, such as, for example, aluminum monostearate,gelatin or combinations thereof.

L. Combinational Therapy

Certain aspects of the present invention pertain to compositionscomprising a chelator that is conjugated to a targeting ligand that is atherapeutic moiety. In other embodiments, the chelator includes an aminoacid sequence that is a therapeutic amino acid sequence.

These compositions can be applied in the treatment of diseases, such ascancer and cardiovascular disease, along with another agent or therapymethod. Treatment with these compositions of the present invention mayprecede or follow the other therapy method by intervals ranging fromminutes to weeks. In embodiments where another agent is administered,one would generally ensure that a significant period of time did notexpire between the time of each delivery, such that the agents wouldstill be able to exert an advantageously combined effect on the cell.For example, it is contemplated that one may administer two, three,three or more doses of one agent substantially simultaneously (i.e.,within less than about a minute) with the compositions of the presentinvention. In other aspects, a therapeutic agent or method may beadministered within about 1 minute to about 48 hours or more prior toand/or after administering a therapeutic amount of a composition of thepresent invention, or prior to and/or after any amount of time not setforth herein. In certain other embodiments, a composition of the presentinvention may be administered within of from about 1 day to about 21days prior to and/or after administering another therapeutic modality,such as surgery or gene therapy. In some situations, it may be desirableto extend the time period for treatment significantly, however, whereseveral weeks (e.g., about 1 to 8 weeks or more) lapse between therespective administrations.

Various combinations may be employed, as demonstrated below, wherein aconjugate of the present invention is designated “A” and the secondaryagent, which can be any other therapeutic agent or method, is “B”:

A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/BB/A/B/B B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/AB/B/A/A B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/A A/A/B/A

Administration of the compositions of the present invention to a patientwill follow general protocols for the administration ofchemotherapeutics, taking into account the toxicity, if any, of theseagents. It is expected that the treatment cycles would be repeated asnecessary. It also is contemplated that various standard therapies, aswell as surgical intervention, may be applied in combination with thedescribed agent. These therapies include but are not limited toadditional pharmacotherapy (such as chemotherapy for cancer), additionalradiotherapy, immunotherapy, gene therapy and surgery.

1. Chemotherapy

Cancer therapies also include a variety of combination therapies withboth chemical and radiation based treatments. Combination chemotherapiesinclude, for example, cisplatin (CDDP), carboplatin, procarbazine,mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan,chlorambucil, busulfan, nitrosurea, dactinomycin, daunorubicin,doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP16),tamoxifen, raloxifene, estrogen receptor binding agents, taxol,gemcitabien, navelbine, farnesyl-protein transferase inhibitors,transplatinum, 5-fluorouracil, vincristin, vinblastin and methotrexate,or any analog or derivative variant of the foregoing.

2. Radiotherapy

Other factors that cause DNA damage and have been used extensivelyinclude what are commonly known as γ-rays, X-rays, and/or the directeddelivery of radioisotopes to tumor cells. Other forms of DNA damagingfactors are also contemplated such as microwaves and UV-irradiation. Itis most likely that all of these factors effect a broad range of damageon DNA, on the precursors of DNA, on the replication and repair of DNA,and on the assembly and maintenance of chromosomes. Dosage ranges forX-rays range from daily doses of 50 to 200 roentgens for prolongedperiods of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens.Dosage ranges for radioisotopes vary widely, and depend on the half-lifeof the isotope, the strength and type of radiation emitted, and theuptake by the neoplastic cells. The terms “contacted” and “exposed,”when applied to a cell, are used herein to describe the process by whicha therapeutic construct and a chemotherapeutic or radiotherapeutic agentare delivered to a target cell or are placed in direct juxtapositionwith the target cell. To achieve cell killing or stasis, both agents aredelivered to a cell in a combined amount effective to kill the cell orprevent it from dividing.

3. Immunotherapy

Immunotherapeutics, generally, rely on the use of immune effector cellsand molecules to target and destroy cancer cells. The immune effectormay be, for example, an antibody specific for some marker on the surfaceof a tumor cell. The antibody alone may serve as an effector of therapyor it may recruit other cells to actually effect cell killing. Theantibody also may be conjugated to a drug or toxin (chemotherapeutic,radionucleotide, ricin A chain, cholera toxin, pertussis toxin, etc.)and serve merely as a targeting agent. Alternatively, the effector maybe a lymphocyte carrying a surface molecule that interacts, eitherdirectly or indirectly, with a tumor cell target. Various effector cellsinclude cytotoxic T cells and NK cells.

Immunotherapy, thus, could be used as part of a combined therapy, inconjunction with gene therapy. The general approach for combined therapyis discussed below. Generally, the tumor cell must bear some marker thatis amenable to targeting, i.e., is not present on the majority of othercells. Many tumor markers exist and any of these may be suitable fortargeting in the context of the present invention. Common tumor markersinclude carcinoembryonic antigen, prostate specific antigen, urinarytumor associated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72,HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, estrogen receptor, lamininreceptor, erb B and p155.

4. Genes

In yet another embodiment, the secondary treatment is a gene therapy inwhich a therapeutic composition is administered before, after, or at thesame time as the therapeutic agents of the present invention. Deliveryof a therapeutic amount of a composition of the present invention inconjunction with a vector encoding a gene product will have a combinedanti-hyperproliferative effect on target tissues.

5. Surgery

Approximately 60% of persons with cancer will undergo surgery of sometype, which includes preventative, diagnostic or staging, curative andpalliative surgery. Curative surgery is a cancer treatment that may beused in conjunction with other therapies, such as the treatment of thepresent invention, chemotherapy, radiotherapy, hormonal therapy, genetherapy, immunotherapy and/or alternative therapies. Curative surgeryincludes resection in which all or part of cancerous tissue isphysically removed, excised, and/or destroyed. Tumor resection refers tophysical removal of at least part of a tumor. In addition to tumorresection, treatment by surgery includes laser surgery, cryosurgery,electrosurgery, and miscopically controlled surgery (Mohs' surgery). Itis further contemplated that the present invention may be used inconjunction with removal of superficial cancers, precancers, orincidental amounts of normal tissue.

M. Other Embodiments of the Present Invention

In one aspect, the present invention generally pertains to a method ofsynthesizing a chelator, such as EC, comprising at least threefunctional groups, the method comprising obtaining a chelator andeither:

-   -   (a) protecting at least one first functional group of the        chelator with a first protecting agent to generate a firstly        protected chelator; or    -   (b) chelating the chelator to a metal ion to generate a metal        ion-labeled chelator.

Any method of synthesis as described herein, such as this, may takeplace in an organic medium, as described herein. The method may furthercomprise at least one purification step, as described herein. Chelators,functional groups, metal ions and modes of chelation and conjugationthat may be used in the methods of the present invention are familiar tothose of ordinary skill in the art and are described herein. Thechelator may further comprise a spacer as described herein, such asethylene. Such chelators are useful intermediates for the preparation ofchelator-targeting ligand conjugates.

In some embodiments, the method comprises protecting at least one firstfunctional group of the chelator with a first protecting agent togenerate a firstly protected chelator. In certain embodiments, the firstfunctional group is a thiol functional group. In certain embodiments,the first protecting agent is a thiol protecting agent. In furtherembodiments, the thiol protecting agent is selected from a groupconsisting of an alkyl halide, a benzyl halide, a benzoyl halide, asulfonyl halide, a triphenylmethyl halide, a methoxytriphenylmethylhalide and cysteine.

The method may, in some embodiments, comprise protecting a secondfunctional group with a second protecting agent to generate a secondlyprotected chelator. In certain embodiments, the first functional groupcomprises at least one thiol functional group and the second functionalgroup comprises at least one amine functional group. In someembodiments, a thiol functional group is first protected with a thiolprotecting agent and then an amine functional group is protected with anamine protecting agent. In further embodiments, an amine protectingagent is selected from the group consisting of benzylchloroformate,p-nitro-chlorobenzylformate, ethylchloroformate,di-tert-butyl-dicarbonate, triphenylmethyl chloride andmethoxytriphenylmethyl chloride. An example of a chelator that may beprepared comprises ethylenedicysteine, wherein the two thiol groups ofethylenedicysteine are protected with two equivalents of a thiolprotecting agent followed by protection of the two amine groups ofethylenedicysteine with two equivalents of an amine protecting agent.Since thiol groups are more reactive than amine groups, thiol groupswill typically be protected before amine groups are protected.

In other embodiments, the method further comprises removing one or moreprotecting groups from any composition described herein comprising oneor more protecting groups. The protecting groups may be removed, forexample, from the chelator moiety, the targeting ligand moiety, or bothmoieties in one or more steps before or after a chelator-targetingligand conjugate is chelated to a metal ion, as described herein.Protecting groups are described in more detail herein, including theirinstallation and removal.

Any composition of the present invention may be purified via any methodknown to those of skill in the art. Methods of purification aredescribed in more detail herein. In some embodiments, the firstlyprotected chelator is between about 90% and about 99.9% pure. In someembodiments, the secondly protected chelator is between about 90% andabout 99.9% pure.

In some embodiments, methods of the present invention further compriseconjugation of a chelator to a targeting ligand, wherein the targetingligand and/or the chelator comprises at least one functional group toform a chelator-targeting ligand conjugate. In some embodiments, afunctional group of the targeting ligand is protected by at least oneprotecting agent prior to conjugation to the chelator. In someembodiments, at least one functional group is a carboxylic acidfunctional group. In some embodiments, the functional groups of thechelator and the targeting ligand together form a chelate. Chelation ofthe metal ion to the chelator can be by any method known to those ofordinary skill in the art.

A chelator-targeting ligand conjugate of the present invention mayfurther comprise a linker between the chelator and the targeting ligand,as described herein. As mentioned, the targeting ligand may of any typeknown to those of skill in the art, and such ligands are discussed inmore detail herein.

Other general aspects of the present invention contemplate a method ofsynthesizing a metal ion labeled-chelator-targeting ligand conjugate,comprising:

-   -   (a) obtaining a protected chelator comprising at least three        functional groups protected by at least one protecting agent;    -   (b) conjugating the protected chelator to a targeting ligand to        generate a chelator-targeting ligand conjugate;    -   (c) removing at least one protecting group from the        chelator-targeting ligand conjugate;    -   (d) chelating a metal ion to the chelator of the        chelator-targeting ligand conjugate; and    -   (e) removing any remaining protecting groups.

The chelator, protecting agents, functional groups, mode of conjugation,targeting ligand, method of removing a protecting group, mode ofchelation and metal ion may be that of any type described herein. Themethod may take place in an organic medium, as described herein. Themethod may comprise one or more purification steps, as described herein.In some embodiments, at least one functional group of the targetingligand is protected by at least one protecting agent prior toconjugation. In preferred embodiments, three or four atoms of thechelator are available for chelation.

Other general aspects of the present invention contemplate a method ofsynthesizing a metal ion labeled-chelator-targeting ligand conjugatecomprising:

-   -   (a) obtaining a chelator comprising at least three functional        groups;    -   (b) chelating a metal ion to the chelator to generate a metal        ion labeled-chelator;    -   (c) conjugating the metal ion labeled-chelator to a targeting        ligand.

The chelator, functional groups, mode of conjugation, targeting ligand,mode of chelation and metal ion may be that of any type describedherein. The method may take place in an organic medium, as describedherein. The method may comprise on or more purification steps, asdescribed herein. In some embodiments, at least one functional group ofthe targeting ligand is protected by at least one protecting agent priorto conjugation. The method may further comprise the removal of allprotecting groups from the metal ion labeled-chelator-targeting ligandconjugate. The method also contemplates, in certain embodiments, atleast one functional group of the targeting ligand being protected by atleast one protecting agent prior to conjugation.

The present invention also contemplates kits for preparing an imagingagent, a chemotherapeutic agent, or a radio/chemotherapeutic agent,comprising one or more sealed containers, and a predetermined quantityof any composition as described herein in one or more of the sealedcontainers. The present invention also contemplates, in someembodiments, an imaging, chemotherapeutic, or radio/chemotherapeuticagent, comprising any composition as described herein.

In some embodiments, the present invention contemplates a method ofimaging or treating a subject, comprising administering to the subject apharmaceutically effective amount of any composition as describedherein. The subject may be a mammal, such as a human.

N. Methods of Diagnosis, Treatment, or Imaging in a Subject with Knownor Suspected Heart Disease

Embodiments of the present invention also generally pertain to methodsof diagnosis, treatment, or imaging in a subject with known or suspectedheart disease. The subject can be any subject, such as a mammal or avianspecies. The mammal, for example, may be a dog, cat, rat, mouse, orhuman. In preferred embodiments, the subject is a human with known orsuspected cardiovascular disease.

The cardiovascular disease can be any disease of the heart or of a bloodvessel. The blood vessel may be a coronary vessel, or may be a vesselother than a coronary vessel. The vessel may be an artery, vein,arteriole, venule, or capillary.

Examples of cardiovascular diseases include diseases of the heart, suchas myocardial infarction, myocardial ischemia, angina pectoris,congestive heart failure, cardiomyopathy (congenital or acquired),arrhythmia, or valvular heart disease. In particular embodiments, thesubject is known or suspected to have myocardial ischemia.

The subject, for example, may be a patient who presents to a clinic withsigns or symptoms suggestive of myocardial ischemia or myocardialinfarction. Imaging of the heart of the subject to diagnose disease mayinvolve administering to the subject a pharmaceutically effective amountof a metal ion labeled chelator-targeting ligand conjugate synthesizedusing any of the methods set forth herein. Imaging can be performedusing any imaging modality known to those of ordinary skill in the art.In particular embodiments, imaging involves use radionuclide-basedimaging technology, such as PET or SPECT. In particular embodiments, themetal ion-labeled radionuclide-targeting ligand conjugate is99m-Tc-EC-glucosamine. Glucosamine is actively taken up by viablemyocardial tissue. Areas of ischemic myocardium would take up less or noconjugate. Severity of ischemia can be visually assessed or gradeddepending on magnitude of the signal that is measured using any methodknown to those of ordinary skill in the art. In some embodiments,imaging using any of the conjugates set forth herein is performedbefore, during, or after imaging of the heart using a second imagingmodality. For example, the second imaging modality may be thalliumscintigraphy.

Myocardial Perfusion SPECT (MPS) consist of a combination of a stressmodality (exercise or pharmacologic) with rest and stress administrationand imaging of radiopharmaceuticals. Thallium has excellent physiologicproperties for myocardial perfusion imaging. Being highly extractedduring the first pass through the coronary circulation, a linearrelationship between blood flow to viable myocardium and thallium uptakehas been shown during exercise; however, at very high levels of flow, a“roll-off” in uptake occurs. As an unbound potassium analogue, thalliumredistributes over time. Its initial distribution is proportional toregional myocardial perfusion and at equilibrium, the distribution ofthallium is proportional to the regional potassium pool, reflectingviable myocardium. The mechanisms of thallium redistribution aredifferential washout rates between hypoperfused but viable myocardiumand normal zones and wash-in to initially hypoperfused zones. Thewashout rate of thallium is the concentration gradient between themyocardial cell and the blood. There is slower blood clearance ofthallium following resting or low-level exercise injection. Diffuse slowwashout rates, mimicking diffuse ischemia, may be observed in normalpatients who do not achieve adequate levels of stress. Hyperinsulinemicstates slow redistribution, leading to an underestimation of viablemyocardium; thus fasting is recommended prior to and for 4 hrs followingthallium injection. This is why if EC-G is used as an viable agent incombination with thallium it will target the precise area of interestwhich would be the viable area (Angello et al., 1987; Gutman et al.,1983; Pohost et al., 1977).

Imaging using any of the metal ion-labeled chelator-targeting ligandconjugates of the present invention may also be performed in conjunctionwith other diagnostic methods, such as measurement of cardiac isozymes,or cardiac catheterization. The imaging may be performed at variousintervals following onset of symptoms, or can be performed to assess forchanges in myocardial perfusion over time.

O. Examples

The following examples are included to demonstrate certain non-limitingaspects of the invention. It should be appreciated by those of skill inthe art that the techniques disclosed in the examples which followrepresent techniques discovered by the inventor to function well in thepractice of the invention. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

The following figures, chemical structures and synthetic details providecertain compounds of the present invention.

Example 1

Non-Limiting Example of an Organic Synthesis ofN,N-Ethylenedicysteine-Glucosamine (EC-G). See FIG. 1.

Step 1: Synthesis of S,S′-Bis-benzyl-N,N′-ethylenedicysteine (Bz-EC)

Cysteine-HCl (30 g) was dissolved in water (100 mL). To this, 37%formaldehyde (22.3 mL) was added and the reaction mixture was stirredovernight at room temperature. Pyridine (25 mL) was then added and aprecipitate formed. The crystals were separated and washed with ethanol(50 mL), then filtered with a Buchner funnel. The crystals weretriturated with petroleum ether (150 mL), again filtered and dried. Theprecursor, L-thiazolidine-4-carboxylic acid (m.p. 195° C., reported196-197° C.) weighed 23.408 g. The precursor (22 g) was dissolved inliquid ammonia (200 mL) and refluxed. Sodium metal was added until apersistent blue color appeared for 15 min. Ammonium chloride was addedto the blue solution, the solvents were evaporated to dryness. Theresidue was dissolved in water (200 mL) and the pH was adjusted to 2with concentrated HCl. A precipitate was formed, filtered and washedwith water (500 mL). The solid was dried in a calcium chloridedessicator. EC was then prepared 10.7 g (m.p. 237° C., reported 251-253°C.). The structure of EC was confirmed by H-1 and C-13 NMR. EC (2.684 g,10 mmol) was dissolved in 1N NaOH (40 mL). Benzyl chloride (5.063 g, 40mmol) was dissolved in dioxane (30 mL) and stirred. The reaction wasstirred for 30 min. The pH of the solution was adjusted to 2 withconcentrated HCl. The precipitate was filtered and washed with water andrecrystallized from trifluoroacetic acid, yielding 79.0% (3.5454 g),m.p. 227-229° C. (dec.) (reported 229-230° C.). The structure of Bz-ECwas confirmed by H-1 and C-13 NMR.

Step 2: Synthesis of S,S′-Bis-benzyl-N,N′-bis-CBZ ethylenedicysteine(Cbz-Bz-EC)

Bz-EC (2.243 g, 5 mmol) was dissolved in sodium carbonate (1.20 g, 11.2mmol) solution and the pH was adjusted to 10 using 1N NaOH. The finalaqueous volume was 30 mL. Benzyl chloroformate (233 mL, 16.5 mmol) wasdissolved in dioxane (0.75 mL) and stirred. The pH was adjusted to 10 byadding solid Na₂CO₃. The reaction mixture was stirred for 2 hours andextracted with diethyl ether to remove the excess benzyl chloroformate(CBZ). The pH of the aqueous layer was adjusted to 2 with 1N HCl andextracted with ethyl acetate. The organic layer was dried over magnesiumsulfate and the solvent was evaporated. The residue was chromatographedon a silica gel column column eluted with CH₂Cl₂:acetic acid (99:1) toCH₂Cl₂:methanol:acetic acid (94:5:1) to yield the desired product 87.2%(3.127 g). The structure of Cbz-Bz-EC was confirmed by H-1 and C-13 NMR.

Step 3: Synthesis of S,S′-Bis-benzyl-N,N′-bis-CBZethylenedicysteine-glucosamine (tetra acetate) Conjugate(Cbz-Bz-EC-G-4-Ac)

To a stirred flask of dichloromethane (22 mL), Cbz-Bz-EC (2.1467 g, 3mmol) was added. This was followed by dicyclohexylcarbodiimide (DCC)(2.476 g, 12 mmol) and dimethylaminopyridine (1.466 g, 12 mmol).Tetraacetylated glucosamine hydrochloride (2.533 g, 6.6 mmol)(4-Ac-G-HCl) (Oakwood Products Inc., West Columbia, S.C.) was added tothe mixture and stirred until completely dissolved. The structure of4-Ac-G-HCl was confirmed by H-1 and C-13 NMR. The reaction was stirredat room temperature overnight. Water (0.5 mL) was added and the solidwas filtered. The filtrate was dried over magnesium sulfate and thesolvent was evaporated. The product was purified by silica gel columnchromatography using dichloromethane:methanol:acetic acid (9.9:0:0.1) to56.4:3:0.6 as a mobile phase. The product was isolated 66.4% yield(2.7382 g). H-1 and C-13 NMR of Cbz-Bz-EC-G-4-Ac provided confirmationas well as mass spectrometry.

Step 4: Synthesis of N,N′-ethylenedicysteine-glucosamine (EC-G)

Cbz-Bz-EC-G-4-Ac (687.7 mg, 0.5 mmol) was dissolved in liquid ammonia(20 mL) and pieces of sodium (223 mg, 10 mmol) were added. After addingall of the sodium, the reaction mixture sustained a dark blue color for20 minutes. Ammonium chloride (641.9 mg, 12 mmol) was added slowly andthe dark blue color solution turned colorless. The liquid ammonia wasremoved by nitrogen. The residual solid was dissolved in water anddialyzed overnight using MW<500. The crude product weighed 206.7 mg(yield: 70%). H-1 and C-13 NMR of the crude EC-G bis-acetylated compoundwere obtained along with mass spectra. The molecular ion was 861 whichcontains the matrix 187 and parent ion 674 (EC-G bis-acetylated). Themajor ion (100%) was 656 which was from the loss of water. EC-Gbis-acetylated compound (200 mg) was further purified by dissolving insodium carbonate and stirring for 2 hours. The product, EC-G, was thenlyophilized, yielding a weight of 70 mg. H-1 NMR and C-13 NMR of EC-Gwere then obtained. C-13 NMR of EC-G showed 16 major carbon peaks. Themass spectra of EC-G was difficult to obtain due to its hydrophilicityand its tendency to be retained on the mass spectrometry column.However, EC-G bis-acetylated compound is less hydrophilic than EC-G;thus, mass spectra of EC-G bis-acetylated could be obtained. Massspectra of EC-G showed that there was small impurity from EC-Gbis-acetylated compound resulting from incomplete hydrolysis procedure.H-1 and C-13 NMR of EC-G were close to the predicted values of EC-G.Although 10 carbon peaks are expected for the symmetric structure ofEC-G, glucosamine has 12 carbons instead of 6 carbons, suggesting thatglucosamine has two configurations. H-1 NMR experimental values appearedto have a somewhat different profile than the predicted values; however,C-13 NMR experimental values of glucosamine were close to the predictedvalues of glucosamine. Thus, EC-G appears to have two configurations.

Example 2 Non-Limiting Example of an Organic Synthesis of ¹⁸⁷Re-EC-GUsing Re-EC and Protected Glucosamine See FIG. 2

¹⁸⁷Re-EC-G was used as a reference standard for ^(99m)Tc-EC due to thesimilarity in structure and lipophilicity. Synthesis of cold Re-EC-G isshown in FIG. 2. To a stirred ethanol solution, small metal sodium chips(144.8 mg, 6.3 mmol) were added slowly into 10 mL of ethanol in a 50 mLbottle under nitrogen. After the sodium metal dissolved, EC (536.8 mg,2.0 mmol) was added. The reaction mixture was stirred for 1 hour at roomtemperature in order to form EC-Na salt. Triphenylphosphine rheniumchloride (ReOCl₃(PPh₃)₂, 1.8329 g, 2.2 mmol) was added. The olive greencolor of ReOCl₃(PPh₃)₂ changed to a forest green color. The reactionmixture was stirred for 1 hour and then refluxed for 30 min. Thereaction mixture was then filtered and the filtrate was evaporated todryness yielding a gray-purple powder Re-EC (818.4 mg, 80% yield). Thestructure of Re-EC was confirmed by H-1 and C-13 NMR and massspectrometry. Re has two isomeric molecular weights which are 185 and187. Therefore, it distinctively shows two parent ions with 40:60ratios.

To a stirred dimethylformamide (4 mL) solvent, Re-EC (116.9 mg, 0.25mmol) was added followed by 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU)(150 mL, 1.0 mmol). Next, dicyclohexylcarbodiimide (DCC) (123.8 mg, 0.6mmol) was added. The reaction mixture was stirred for 1 hour.Tetraacetylated glucosamine (4-Ac-G-HCl) (184.9 mg, 0.5 mmol) was addedand then the reaction was stirred at room temperature overnight. Water(1 mL) was added and the reaction stirred for additional 1 hour at roomtemperature. The reaction mixture was evaporated under reduced pressure.Water (5 mL) was added, followed by chloroform (5 mL). The water layerwas separated and lyophilized to yield a crude dark-brown solid. Thesolid was purified by column chromatography using Sephadex G-50 to yieldcold Re-EC-G (128.4 mg, 65% yield). The structure of cold Re-EC-G wasconfirmed by H-1 and C-13 NMR and mass spectrometry. Again, theRe-complex distinctively shows two parent ions with 40:60 ratios.

Elemental analysis of cold Re-EC-G showed C₂₀H₃₅N₄O₁₃ReS₂ (C, H, N) withthe calculated value C, 30.41; H, 4.47; N, 7.09; found value C, 30.04;H, 4.93; N, 6.09. H-1 and C-13 NMR of cold Re-EC-G was similar to thepredicted NMR spectrometry. EC-G (5 mg) was labeled with ^(99m)Tc(pertechnetate) (1 mCi) in the presence of tin(II) chloride (0.1 mg).HPLC analysis showed that cold Re-EC-G had a similar retention time tothat of ^(99m)Tc-EC-G.

Example 3 Synthesis of EC-G Using EC and Glucosamine in an AqueousReaction

EC (107 mg, 0.4 mmol) was dissolved in NaHCO₃ (1N, 12 mL). To thiscolorless solution, sulfo-N-hydroxysuccinimide (sulfo-NHS, 173.7 mg, 0.8mmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide-HCl (EDAC)(Aldrich Chemical Co, Milwaukee, Wis.) (153.4 mg, 0.8 mmol) were added.D-Glucosamine hydrochloride salt (Sigma Chemical Co., St Louis, Mo.)(345 mg, 1.6 mmol) was then added. A pH of 8 was measured. The mixturewas stirred at room temperature for 16 hours and then dialyzed for 24hours using Spectra/POR molecular porous membrane with cut-off at 500(Spectrum Medical Industries Inc., Houston, Tex.). After dialysis, theproduct was filtered by a 0.45 μm Nylon-filter and then freeze-driedusing a lyophilizer (Labconco, Kansas City, Mo.). The crude productweighed 300-400 mg. H-1 NMR of EC-G showed similar patterns; however, itappears that the mixture is not as pure when compared to the organicEC-G. Elemental analysis showed EC-G purity was 63-77% using differentreaction ratios between EC and glucosamine. Prep-HPLC (7.8×300 mm C-18column, Waters) (flow rate: 0.5 mL/min, 100% water, UV 235 nm) was usedto purify the crude product, 180-240 mg (yield 60%). H-1 and C-13 NMR ofEC-G after prep-HPLC showed additional peaks suggesting impurities frommono EC-G or EC-glucosamine, sulfo-NHS and EDAC. Prep-HPLC purificationof the raw EC-G yielded some incremental improvement to the chemicalpurity; however, when the raw EC-G is labeled with ^(99m)Tc in thepresence of tin(II) chloride, a greater than 95% radiochemical purity of^(99m)Tc-EC-G can be achieved using gluconate as a transchelator (asshown in radio-TLC and HPLC analysis).

Example 4 Cellular Uptake Study Comparing Products Synthesized Via theAqueous Method and the Organic Method

To further validate EC-G biological activity, in vitro cell cultureassays were performed. Briefly, the cellular uptake was determined intumor cells (50,000 cells/well) incubated with ^(99m)Tc-EC-G (2μCi/well) at various time intervals. The cellular uptake assay showed nomarked difference between raw (unpurified) EC-G and prep-HPLC purifiedEC-G (FIG. 4) In vitro stability studies were determined either usingcell culture or dissolving EC-G in water. There was a 10-15% decrease incellular uptake using ^(99m)Tc-EC-G after 2-4 weeks. The useful life ofEC-G in water appears to be 17.26 days. In vivo imaging studies showedno marked difference between EC-G synthesized from aqueous and organicreactions.

Example 5 Synthesis of Cold Re-EC-G Using Re-EC and Glucosamine in anAqueous Reaction

Re-EC (255.8 mg, 0.5 mmol) (from Example 2) was dissolved in NaOH (1N,4.5 mL). Added to this dark-purple color solution were sulfo-NHS (217.1mg, 1 mmol) and D-glucosamine hydrochloride salt (Sigma Chemical Co.,St. Louis, Mo.) (431.3 mg, 2 mmol).1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide-HCl (EDAC) (AldrichChemical Co., Milwaukee, Wis.) (191.7 mg, 1 mmol) was then added. The pHmeasured greater than 8. The mixture was stirred at room temperature for16 hours. The mixture was dialyzed for 24 hours using Spectra/PORmolecular porous membrane with cut-off at 500 (Spectrum MedicalIndustries Inc., Houston, Tex.). After dialysis, the product wasfiltered and then freeze-dried using a lyophilizer (Labconco, KansasCity, Mo.). The crude product weighed 276 mg. H-1 NMR of aqueous Re-EC-Gshowed a similar pattern; however, there appears to be some evidence ofimpurities when compared to the organic Re-EC-G compound. HPLC analysisof the organic cold Re-EC-G compound showed one peak at 272 nm; however,aqueous cold Re-EC-G had two peaks. One of the peaks in the aqueous coldRe-EC-G corresponds to the organic cold Re-EC-G compound (peaks 12.216and 12.375, respectively). The remaining peaks were sulfo-NHS and otherminor impurities.

Example 6 Quantitative Analysis of Glucosamine (Active PharmaceuticalIngredient)

D-Glucosamine was derivatized for colorimetric assays. Briefly, to asolution of D-glucosamine hydrochloride (25 g, 0.12 mol) in a freshlyprepared aqueous solution of 1N NaOH (120 mL) under stirring was addedp-anisaldehyde (17 mL, 0.14 mol). After 30 min., crystallization beganand the mixture was refrigerated overnight. The precipitated product wasthen filtered and washed with cold water (60 mL), followed by a mixtureof EtOH-Et₂O (1:1) to give2-deoxy-2-[p-methoxybenzylidene(amino)]-D-glucopyranose(D-glucosamine-anisaldehyde, 32.9408 g, 110.8 mmol, 95.5% yield) m.p.165-166° C. H-1 NMR confirmed the structure.

Raw EC-G (50 mg) was hydrolyzed using 1N NaOH. Anisaldehyde was added tothe reaction solution. After 2 hours, the reaction mixture was extractedwith chloroform. The chloroform layer, which contained unreactedanisaldehyde, was evaporated under nitrogen. The reacted anisaldehydeweight was used to determine the amount of glucosamine in theD-glucosamine-anisaldehyde adduct.

Example 7

Quantitative Analysis of EC in EC-G

Raw EC-G (50 mg) was hydrolyzed using 1N NaOH. Benzyl chloride wasdissolved in dioxane (30 mL) and then added in to the stirred mixture.The reaction was stirred for 2 hours and then extracted with chloroform.The chloroform layer, which contained unreacted benzyl chloride, wasevaporated under nitrogen. The reacted benzyl chloride weight was usedto determine the amount of EC in EC-G (Table 3).

Example 8 Quantitative Analysis of Sulfo-NHS and EDAC in EC-G

A standard curve of sulfo-NHS was generated at UV 272 nm. Raw EC-G wasdissolved in water. From the standard curve, the amount of sulfo-NHS inEC-G was determined at UV 272 nm. The amount of1-ethyl-3-(3-dimethylaminopropyl)carbodiimide-HCl (EDAC) was calculatedby subtracting EC, glucosamine and sulfo-NHS from total EC-G weightshown in Table 3.

Example 9 Quantitative Analysis of Glucose Phosphorylation Assay

An in vitro hexokinase assay was used to assess the glucosephosphorylation process of EC-G. Using a kit (Sigma Chemical Company,MO), fluorodeoxyglucose (FDG, 1.0 mg), EC-G (1.0 mg), D-glucosamine (G,1.0 mg) and D-glucose (2.5 mg) were dissolved in 1 mL (EC-G, G) or 2.5mL (D-glucose) of water. From there, 200 μL was removed and diluted in2.5 mL of water. A 100 μL aliquot was then removed and combined insolution with 900 μL of Infinity™ Glucose Reagent and incubated at 37°C. for three min. The phosphorylated glucose and NADH were then assayedat a wavelength of 340 nm. The peaks of FDG (340 and 347 nm), glucose(301 and 342 nm), EC-G (303 and 342 nm) and G (302 and 342 nm) wereobtained.

Example 10 Chemical Identity Assay of Glucosamine (Active PharmaceuticalIngredient) in EC-G (Synthesized from the Aqueous Reaction)

A colorimetric assay was used to determine the amount of glucosamine. Asolution of copper sulfate (6.93 g in 100 mL water) and sodium potassiumtartrate (34.6 g in 100 mL water containing 10 g NaOH) was prepared.EC-G (25 mg) and glucosamine (standard) were added with basic coppertartrate solution until no visualization of copper oxide red precipitateexisted. The amount of glucosamine in EC-G was 8.7 mg (35% w/w)determined from titration volume (Table 3).

Alternatively, as described in Example 5, D-glucosamine hydrochloride(25 g, 0.12 mol) was added to a freshly prepared aqueous solution of 1NNaOH (120 mL) under stirring and then p-anisaldehyde (17 mL, 0.14 mol)was added to the mixture. After 30 min., the crystallization began andthe mixture was refrigerated overnight. The precipitated product wasfiltered and washed with cold water (60 mL), followed by a mixture ofEtOH-Et₂O (1:1) to yield2-deoxy-2-[p-methoxybenzylidene(amino)]-D-glucopyranose(D-glucosamine-anisaldehyde, 32.9408 g, 110.8 mmol, 95.5% yield) m.p.165-166° C. Raw EC-G (50 mg) was hydrolyzed using 1N NaOH. Anisaldehydewas added to the reaction solution. After 30 min., the crystallizationbegan and the mixture was refrigerated overnight. The precipitatedproduct was filtered and washed with cold water and the melting pointwas determined to be 165-166° C. (containing 18 mg glucosamine).

TABLE 3 Qualitative Analysis of Glucosamine and EC in EC-G (synthesizedfrom the aqueous reaction) Theoretical Value Percentage (weight/weight)Compound Molecular Weight (100%) (65%) EC-G 591 EC 268 Glucosamine (G)179 EC in EC-G 39% (234/591) 25% G in EC-G 60% (356/591) 39%Experimental Value Percentage Compound (weight/weight) Method EC in EC-G30% colorimetric G in EC-G 35% colorimetric Sulfo-NHS in EC-G 34% UV(268 nm) EDAC  1% calculation

Example 11 Chemical Identity Assay of Ethylenedicysteine (Chelator) inEC-G (Synthesized from the Aqueous Reaction)

Two methods were used to determine the purity of EC-G. In the firstmethod, a colorimetric assay was used to determine the amount of EC. Asolution of iodine (0.1 mol/L) (13 g along with 36 g KI in 1000 mLwater) was prepared and EC-G (25.2 mg) and EC (25 mg) (standard) wereadded to the iodine solution. In the standard EC, a pale white solid wasprecipitated, but no precipitate was noted in the EC-G. A titrationmethod was used (yellowish color (persists more than 5 min.)) todetermine the amount of EC in the EC-G. Each 1 mL of iodine solutionthat was used equals 13.4 mg of EC. The amount of EC in the EC-G was 7.6mg (30.2% w/w).

In the second method, measurement the melting point of a thiol-EC-Gadduct was performed. Example 1 outlined the synthesis ofS,S′-Bis-benzyl-N,N′-ethylenedicysteine (Bz-EC). Briefly, EC (2.684 g,10 mmol) was dissolved in 1N NaOH (40 mL). Benzyl chloride (5.063 g, 40mmol) was dissolved in dioxane (30 mL) and added to a stirred mixture.After 30 min., the pH of the solution was adjusted to 2 withconcentrated HCl. The precipitate was filtered and washed with water andrecrystallized from trifluoroacetic acid. The yield was 79.0% (3.5454g), m.p. 227-229° C. (dec.) (reported 229-230° C.). Raw EC-G (50 mg) wasthen hydrolyzed using 1N NaOH, and benzyl chloride (40 mg) was added.The reaction mixture was stirred for 30 min. The pH of the solution wasadjusted to 2 with concentrated HCl. The precipitate was filtered andwashed with water to give EC-benzyl adduct, m.p. 227-229° C. (containingEC 16 mg).

Example 12 Chemical Identity Assay of Sulfo-N-Hydroxysuccinimide(Sulfo-NHS) in EC-G (Synthesized from the Aqueous Reaction)

The assay for N-hydroxysulfosuccinimide (sulfo-NHS) was determined by UV(268 nm). A standard curve of sulfo-NHS was produced at UV 268 nm. Underthis UV absorbance, poor absorbance was observed for EC-G and EDAC. RawEC-G (50 μg/mL) was dissolved in water and the absorbance was measuredat 268 nm. The estimated sulfo-NHS was 35±5% (w/w).

Example 13 Radiochemical Purity and Identity Assay

Thin-layered chromatography (TLC) and high performance liquidchromatography (HPLC) were used to determine radiochemical identity. Forthe TLC assay, both aqueous and organic synthesized EC-G were labeledwith ^(99m)Tc and spotted on a TLC strip impregnated with silica gelcolumn (ITLC-SG) and scanned using a radio-TLC scanner. The retentionfactor (Rf) values of ^(99m)Tc-EC-G (from the aqueous synthesis) and thereference standard (^(99m)Tc-EC-G from the organic synthesis) were 0.8(determined by ammonium acetate (1M):methanol; 4:1) or saline. For theHPLC assay, the chemical purity of the organic and aqueous synthesizedEC-G were 95.64% and 90.52%, respectively. EC-G synthesized from theorganic reaction was more pure than EC-G synthesized from the aqueousreaction. Both the organic and aqueous synthesized EC-G were labeledwith ^(99m)Tc and loaded (20 μL, 1 mg/mL EC-G) on a C-18 reverse phasecolumn (Waters, semi-prep, 7.8×300 mm). The retention time (Rt) valuesof ^(99m)Tc-EC-G and cold Re-EC-G (the reference standard from theorganic synthesis) were between 11.7-13.5 min. (determined by 100% water@ 0.5 mL/min, UV at 210 nm). Both the organic and aqueous synthesized^(99m)Tc-EC-G were detected by UV wavelength (210 nm) and the matchedradioactive detector findings were within the above stated ranges. Invitro cell culture assays showed that Re-EC-G produced a dose responsecurve (FIG. 3) and was effective against human lymphoma cells.

Summary:

-   -   The radiochemical purity of the ^(99m)Tc-EC-G measured by HPLC        and TLC is greater than 95% for the aqueous synthesized EC-G,        which closely approximates the radiochemical purity for the        organic synthesized EC-G.    -   The chemical purity of the unlabeled aqueous EC-G measured by        colorimetric and elemental analysis falls in the range of        60-70%. All impurities contained in the EC-G compound (whether        the aqueous or organic synthesis) have been clearly identified        through calorimetric assays and UV spectrometry as glucosamine        (35%), EC (30%), sulfo-NHS (34%) and EDAC (1%) on a % w/w basis.    -   When measured by HPLC at UV 210 nm, the chemical purity of the        unlabeled aqueous EC-G compares very favorably to the unlabeled        organic EC-G at 90.52% vs. 95.64%, respectively.

Retention time of the aqueous ^(99m)Tc-EC-G is in the range of coldRe-EC-G measured by HPLC at 272 nm.

-   -   NMR (¹H, ¹³C) of aqueous EC-G is in the range of cold Re-EC-G.    -   Unlabeled organic EC-G, labeled organic EC-G and cold Re-EC-G        are used as reference standards.    -   Biologic assays (in vitro uptake and in vivo imaging) showed no        marked difference between aqueous and organic synthesized EC-G.

Example 14 Purity Analysis of ⁶⁸Ga-EC-G

⁶⁸Ga-EC-G synthesized by both organic and aqueous means were analyzedvia radio-TLC. FIG. 6 shows the improved purity of the organic product(a) over the aqueous product (b). FIG. 7 represents purificationperformed on a C-18 column (Puresil, 4.6×150 mm, Waters, Milford, Mass.)and eluted with water using a flow rate of 0.5 ml/min. Detection wasperformed via UV and NaI.

Example 15 Stability Analysis of ⁶⁸Ga-EC-G

FIG. 8 depicts the results of a study of the stability of ⁶⁸Ga-EC-G indog serum as shown by radio-TLC. 100 μL ⁶⁸Ga-EC-G (0.7 mg/0.7 ml, pH7.5, 865 μCi) were added to 100 μL dog serum and incubated for 0, 30, 60and 120 minutes. Next, 200 μL MeOH were added to each sample andvortexed before elution using a system comprisingpyridine:EtOH:water=1:2:4; Whatman #1 paper. (a) ⁶⁸Ga-EC-G (0.7 mg/0.7ml, pH 7.5, 865 μCi); (b) 100 μL ⁶⁸Ga-EC-G in 100 μL dog serum, time=0;(c) time=30 min.; (d) time=60 min.; (e) time=120 min.; (f) ⁶⁸Ga-EC-BSA.

FIG. 9 depicts the results of a study of the stability of ⁶⁸Ga-EC-G indog serum as analyzed in a protein binding assay. A control sample wasincubated with ⁶⁸Ga-EC-bovine serum albumin (BSA) in dog serum. 100 μL⁶⁸Ga-EC-G (0.7 mg/0.7 ml, pH 7.5, 865 μCi) were added to 100 μL dogserum and incubated for 0, 30, 60 and 120 minutes, the activity counted,then 200 μL MeOH was added and the sample vortexed, centrifuged for 1minute, and then supernatant and precipitate were each counted. Thecounts determined in the precipitate are indicative of the degree ofbinding between ⁶⁸Ga-EC-G and proteins in the dog serum.

The protein binding rate increased from 18.6% to 51.5% after 2 hrs,suggesting the targeting potential of ⁶⁸Ga-EC-G.

Example 16 In Vitro Update Study of 68Ga-Labeled Compounds in BreastCancer Cell Line 13762

FIG. 10 depicts results from an in vitro uptake study of ⁶⁸Ga-labeledcompounds in breast cancer cell line 13762. Cellular uptake of ⁶⁸Ga-ECand ⁶⁸Ga-EC-G in 13762 cells (1 μCi/50,000 cells per well). Cellularuptake of ⁶⁸Ga-EC-G was significantly (p<0.01) higher than control⁶⁸Ga-EC at 0.5-2 hrs.

Example 17 Imaging of Cardiovascular Disease

FIG. 11 shows planar scintigraphy images of a ^(99m)Tc-EC-ESMOLOLderivative (300 μCi/rat) in breast tumor-bearing rats. The numbers areheart/upper mediastinum (H/UM) count density (count/pixel) ratios at15-45 minutes. The line profile in FIG. 11 shows a high cardiac regioncount/pixels ratio in comparison to laterally located tissues. Theseresults demonstrate that ^(99m)Tc-EC-ESMOLOL is surprisingly effectiveat imaging the cardiac region. FIG. 12 shows ⁶⁸Ga-EC-TML PET imagingresults in a New Zealand white rabbit. A rabbit was administered⁶⁸Ga-EC-trimethyl lysine (EC-TML). PET coronal images were acquired at45 minutes after injection of 0.66 mCi of ⁶⁸Ga-EC-TML (dorsal to ventralorder). High uptake in the heart was noticed, suggesting EC-TML wasinvolved in fatty acid metabolism.

Example 18 Non-Limiting Example of Organic Synthesis of Ec-G Via anEC-Benzhydrol-Cbz-Glucosamine Intermediate (see FIG. 13)

EC-Benzhydrol-Cbz-Glucosamine can be dissolved in ethyl acetate andprecipitated out by adding MTBE or n-Hexane. This was envisioned as amethod of obtaining pure a penultimate species in a method of obtainingEC-G. The purity (HPLC) of EC-Benzhydrol-Cbz-Glucosamine before thistrituration treatment was about 64%. After trituration, the purity wasabout 68% (MTBE) or 65%-80% (n-Hexane). Another envisioned method forpurifying the product is through use of a biotage cartridge, as sincethe silica gel in these cartridges is more active than flash gradesilica gel.

Other purification techniques and procedures were also attempted usingdifferent solvent systems as an alternative to chromatography, theresults of which are shown in Table 4 below. Precipitation was attemptedin different solvent systems. The EC-Benzhydrol-Cbz was dissolved in aselected solvent (A), and slowly charged to a larger volume ofco-solvent (B). However, the results did not indicate this approachwould be as effective as other methods, as the purity changes werenegligible. Triturations were also attempted using the selected solventsystems in various ratios for precipitation. The results for thetriturations also suggest the material is not pure enough for certainapplications. Column chromatography was also attempted, and theconditions were modified from the previous week (15:1 silica:crude,loaded dry on silica). This method did allow for moderate clean up ofthe material (from 55A % to 75A %).

TABLE 4 EC-Benzyhydrol-Cbz Purification by Precipitation and TriturationSolvent A Solvent B Precipitation Result Trituration Result EthylAcetate Hexane Sticky solid Oil Methanol Water Sticky oil Oil DCM HexaneSticky oil Oil Ethanol Water Sticky oil Oil

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and methods and in the steps or in the sequence of steps ofthe method described herein without departing from the concept, spiritand scope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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What is claimed is:
 1. A method of synthesizing a chelator-targetingligand conjugate, comprising: (a) conjugating, in an organic medium, aprotected ethylenedicysteine of the following formula:

to at least one unprotected glucosamine, wherein: A and D are each aprotected thiol; B and C are each a tertiary amine, wherein the tertiaryamine is a protected secondary amine; and the conjugation is via anamide bond formed between a COOH group of the protectedethylenedicysteine and the amino group of the unprotected glucosamine toform a protected ethylenedicysteine glucosamine; and (b) removing eachprotecting group, in one or more steps, from the protectedethylenedicysteine glucosamine to form a chelator-targeting ligandconjugate, wherein the chelator-targeting ligand conjugate isethylenedicysteine glucosamine (EC-G), wherein the purity of thechelator-targeting ligand conjugate is between about 80% (w/w) and about99.9% (w/w), as measured by HPLC using ELSD detection.
 2. The method ofclaim 1, wherein the organic medium comprises a polar solvent.
 3. Themethod of claim 1, wherein the organic medium is dimethylformamide,dimethylsulfoxide, dioxane, methanol, ethanol, hexane, methylenechloride, acetonitrile, tetrahydrofuran, or a mixture thereof.
 4. Themethod of claim 1, further comprising at least one purification step,wherein the purification step is silica gel column chromatography, HPLC,or a combination thereof.
 5. The method of claim 4, wherein the purityof the chelator-targeting ligand conjugate is between about 85% (w/w)and about 99.9% (w/w), as measured by HPLC using ELSD detection.
 6. Themethod of claim 5, wherein the purity of the chelator-targeting ligandconjugate is between about 90% (w/w) and about 99.9% (w/w), as measuredby HPLC using ELSD detection.
 7. The method of claim 1, furthercomprising chelating a metal ion to the chelator-targeting ligandconjugate to generate a metal ion labeled-chelator-targeting ligandconjugate.
 8. The method of claim 7, wherein the purity of the metal ionlabeled-chelator-targeting ligand conjugate is between about 80% w/w andabout 99.9% w/w, as measured by HPLC using ELSD detection.
 9. The methodof claim 8, wherein the purity of the metal ionlabeled-chelator-targeting ligand conjugate is between about 85% w/w andabout 99.9% w/w, as measured by HPLC using ELSD detection.
 10. Themethod of claim 9, wherein the purity of the metal ionlabeled-chelator-targeting ligand conjugate is between about 90% w/w andabout 99.9% w/w, as measured by HPLC using ELSD detection.
 11. Themethod of claim 7, wherein the metal ion is selected from the groupconsisting of a technetium ion, a copper ion, an indium ion, a thalliumion, a gallium ion, an arsenic ion, a rhenium ion, a holmium ion, ayttrium ion, a samarium ion, a selenium ion, a strontium ion, agadolinium ion, a bismuth ion, an iron ion, a manganese ion, a luteciumion, a cobalt ion, a platinum ion, a calcium ion and a rhodium ion. 12.The method of claim 7, wherein the metal ion is ¹⁸⁷Re, ^(99m)Tc,platinum, or ¹⁸⁸Re.
 13. The method of claim 7, wherein the metal ion isa radionuclide.
 14. The method of claim 13, wherein the radionuclide isselected from the group consisting of ^(99m)Tc, ¹⁸⁸Re, ¹⁸⁶Re, ¹⁵³Sm,¹⁶⁶Ho, ⁹⁰Y, ⁸⁹Sr, ⁶⁷Ga, ⁶⁸Ga, ¹¹¹In, ¹⁸³Gd, ⁵⁹Fe, ²²⁵Ac, ²¹²Bi, ²¹¹At,⁴⁵Ti, ⁶⁰Cu, ⁶¹Cu, ⁶⁷Cu, ⁶⁴Cu and ⁶²Cu.
 15. The method of claim 14,further comprising the addition of a reducing agent.
 16. The method ofclaim 1, wherein the protected thiol is protected using a thiolprotecting agent selected from a group consisting of an alkyl halide, abenzyl halide, a benzoyl halide, a sulfonyl halide, benzhydrol, atriphenylmethyl halide, a methoxytriphenylmethyl halide and cysteine.17. The method of claim 1, wherein the protected tertiary amine isprotected using an amine protecting agent selected from the groupconsisting of benzylchloroformate, p-nitro-chlorobenzylformate,ethylchloroformate, di-tert-butyl-dicarbonate, triphenylmethyl chlorideand methoxytriphenylmethyl chloride.
 18. The method of claim 1, whereinthe protected ethylenedicysteine glucosamine isEC-Benzhydrol-Cbz-Glucosamine.